Inbred corn line PHACE

ABSTRACT

A novel inbred maize line designated PHACE and seed, plants and plant parts thereof. Methods for producing a maize plant that comprise crossing inbred maize line PHACE with another maize plant. Methods for producing a maize plant containing in its genetic material one or more traits introgressed into PHACE through backcross conversion and/or transformation, and to the maize seed, plant and plant part produced thereby. Hybrid maize seed, plant or plant part produced by crossing the inbred line PHACE or an introgressed trait conversion of PHACE with another maize line. Inbred maize lines derived from inbred maize line PHACE, methods for producing other inbred maize lines derived from inbred maize line PHACE and the inbred maize lines and their parts derived by the use of those methods.

This application is a continuation of application Ser. No. 10/769,188,now U.S. Pat. No. 6,958,438 filed Jan. 30, 2004. The instant applicationincorporates herein the disclosure of application Ser. No. 10/768,188,in its entirety, by reference.

FIELD OF THE INVENTION

This invention relates generally to the field of maize breeding,specifically relating to an inbred maize line designated PHACE.

BACKGROUND OF THE INVENTION

The goal of plant breeding is to combine, in a single variety or hybrid,various desirable traits. For field crops, these traits may includeresistance to diseases and insects, tolerance to heat and drought,reducing the time to crop maturity, greater yield, and better agronomicquality. With mechanical harvesting of many crops, uniformity of plantcharacteristics such as germination, stand establishment, growth rate,maturity, plant height and ear height, is important. Traditional plantbreeding is an important tool in developing new and improved commercialcrops.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel inbred maize line,designated PHACE and processes for making PHACE. This invention relatesto seed of inbred maize line PHACE, to the plants of inbred maize linePHACE, to plant parts of inbred maize line PHACE, and to processes formaking a maize plant that comprise crossing inbred maize line PHACE withanother maize plant. This invention also relates to processes for makinga maize plant containing in its genetic material one or more traitsintrogressed into PHACE through backcross conversion and/ortransformation, and to the maize seed, plant and plant part produced bysuch introgression. This invention further relates to a hybrid maizeseed, plant or plant part produced by crossing the inbred line PHACE oran introgressed trait conversion of PHACE with another maize line. Thisinvention also relates to inbred maize lines derived from inbred maizeline PHACE, to processes for making other inbred maize lines derivedfrom inbred maize line PHACE and to the inbred maize lines and theirparts derived by the use of those processes.

Definitions

Certain definitions used in the specification are provided below. Alsoin the examples that follow, a number of terms are used herein. In orderto provide a clear and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided. NOTE: ABS is in absolute terms and % MN ispercent of the mean for the experiments in which the inbred or hybridwas grown. PCT designates that the trait is calculated as a percentage.% NOT designates the percentage of plants that did not exhibit a trait.For example, STKLDG % NOT is the percentage of plants in a plot thatwere not stalk lodged. These designators will follow the descriptors todenote how the values are to be interpreted.

ABTSTK=ARTIFICIAL BRITTLE STALK. A count of the number of “snapped”plants per plot following machine snapping. A snapped plant has itsstalk completely snapped at a node between the base of the plant and thenode above the ear. Expressed as percent of plants that did not snap.

ALLELE. Any of one or more alternative forms of a genetic sequence.Typically, in a diploid cell or organism, the two alleles of a givensequence typically occupy corresponding loci on a pair of homologouschromosomes.

ANT ROT=ANTHRACNOSE STALK ROT (Colletotrichum graminicola). A 1 to 9visual rating indicating the resistance to Anthracnose Stalk Rot. Ahigher score indicates a higher resistance.

BACKCROSSING. Process in which a breeder crosses a hybrid progeny lineback to one of the parental genotypes one or more times.

BARPLT=BARREN PLANTS. The percent of plants per plot that were notbarren (lack ears).

BREEDING. The genetic manipulation of living organisms.

BREEDING CROSS. A cross to introduce new genetic material into a plantfor the development of a new variety. For example, one could cross plantA with plant B, wherein plant B would be genetically different fromplant A. After the breeding cross, the resulting F1 plants could then beselfed or sibbed for one, two, three or more times (F1, F2, F3, etc.)until a new inbred variety is developed. For clarification, such newinbred varieties would be within a pedigree distance of one breedingcross of plants A and B. The process described above would be referredto as one breeding cycle.

BRTSTK=BRITTLE STALKS. This is a measure of the stalk breakage near thetime of pollination, and is an indication of whether a hybrid or inbredwould snap or break near the time of flowering under severe winds. Dataare presented as percentage of plants that did not snap.

CELL. Cell as used herein includes a plant cell, whether isolated, intissue culture or incorporated in a plant or plant part.

CLDTST=COLD TEST. The percent of plants that germinate under cold testconditions.

CLN=CORN LETHAL NECROSIS. Synergistic interaction of maize chloroticmottle virus (MCMV) in combination with either maize dwarf mosaic virus(MDMV-A or MDMV-B) or wheat streak mosaic virus (WSMV). A 1 to 9 visualrating indicating the resistance to Corn Lethal Necrosis. A higher scoreindicates a higher resistance.

COMRST=COMMON RUST (Puccinia sorghl). A 1 to 9 visual rating indicatingthe resistance to Common Rust. A higher score indicates a higherresistance.

CROSS POLLINATION. A plant is cross pollinated if the pollen comes froma flower on a different plant from a different family or line. Crosspollination excludes sib and self pollination.

CROSS. As used herein, the term “cross” or “crossing” can refer to asimple X by Y cross, or the process of backcrossing, depending on thecontext.

D/D=DRYDOWN. This represents the relative rate at which a hybrid willreach acceptable harvest moisture compared to other hybrids on a 1 to 9rating scale. A high score indicates a hybrid that dries relatively fastwhile a low score indicates a hybrid that dries slowly.

DIPERS=DIPLODIA EAR MOLD SCORES (Diplodia maydis and Diplodiamacrospora). A 1 to 9 visual rating indicating the resistance toDiplodia Ear Mold. A higher score indicates a higher resistance.

DIPLOID PLANT PART. Refers to a plant part or cell that has the samediploid genotype as PHACE.

DIPROT=DIPLODIA STALK ROT SCORE. Score of stalk rot severity due toDiplodia (Diplodia maydis). Expressed as a 1 to 9 score with 9 beinghighly resistant.

DRPEAR=DROPPED EARS. A measure of the number of dropped ears per plotand represents the percentage of plants that did not drop ears prior toharvest.

D/T=DROUGHT TOLERANCE. This represents a 1 to 9 rating for droughttolerance, and is based on data obtained under stress conditions. A highscore indicates good drought tolerance and a low score indicates poordrought tolerance.

EARHT=EAR HEIGHT. The ear height is a measure from the ground to thehighest placed developed ear node attachment and is measured incentimeters.

EARMLD=GENERAL EAR MOLD. Visual rating (1 to 9 score) where a “1” isvery susceptible and a “9” is very resistant. This is based on overallrating for ear mold of mature ears without determining the specific moldorganism, and may not be predictive for a specific ear mold.

EARSZ=EAR SIZE. A 1 to 9 visual rating of ear size. The higher therating the larger the ear size.

EBTSTK=EARLY BRITTLE STALK. A count of the number of “snapped” plantsper plot following severe winds when the corn plant is experiencing veryrapid vegetative growth in the V5–V8 stage. Expressed as percent ofplants that did not snap.

ECB1LF=EUROPEAN CORN BORER FIRST GENERATION LEAF FEEDING (Ostrinianubilalis). A 1 to 9 visual rating indicating the resistance topreflowering leaf feeding by first generation European Corn Borer. Ahigher score indicates a higher resistance.

ECB2IT=EUROPEAN CORN BORER SECOND GENERATION INCHES OF TUNNELING(Ostrinia nubilalis). Average inches of tunneling per plant in thestalk.

ECB2SC=EUROPEAN CORN BORER SECOND GENERATION (Ostrinia nubilalis). A 1to 9 visual rating indicating post flowering degree of stalk breakageand other evidence of feeding by European Corn Borer, Second Generation.A higher score indicates a higher resistance.

ECBDPE=EUROPEAN CORN BORER DROPPED EARS (Ostrinia nubilalis). Droppedears due to European Corn Borer. Percentage of plants that did not dropears under second generation corn borer infestation.

EGRWTH=EARLY GROWTH. This is a measure of the relative height and sizeof a corn seedling at the 2–4 leaf stage of growth. This is a visualrating (1 to 9), with 1 being weak or slow growth, 5 being averagegrowth and 9 being strong growth. Taller plants, wider leaves, moregreen mass and darker color constitute higher score.

ELITE INBRED. An inbred that contributed desirable qualities when usedto produce commercial hybrids. An elite inbred may also be used infurther breeding for the purpose of developing further improvedvarieties.

ERTLDG=EARLY ROOT LODGING. Early root lodging is the percentage ofplants that do not root lodge prior to or around anthesis; plants thatlean from the vertical axis at an approximately 30 degree angle orgreater would be counted as root lodged.

ERTLPN=EARLY ROOT LODGING. An estimate of the percentage of plants thatdo not root lodge prior to or around anthesis; plants that lean from thevertical axis at an approximately 30 degree angle or greater would beconsidered as root lodged.

ERTLSC=EARLY ROOT LODGING SCORE. Score for severity of plants that leanfrom a vertical axis at an approximate 30 degree angle or greater whichtypically results from strong winds prior to or around floweringrecorded within 2 weeks of a wind event. Expressed as a 1 to 9 scorewith 9 being no lodging.

ESTCNT=EARLY STAND COUNT. This is a measure of the stand establishmentin the spring and represents the number of plants that emerge on perplot basis for the inbred or hybrid.

EYESPT=EYE SPOT (Kabatiella zeae or Aureobasidium zeae). A 1 to 9 visualrating indicating the resistance to Eye Spot. A higher score indicates ahigher resistance.

FUSERS=FUSARIUM EAR ROT SCORE (Fusarium moniliforme or Fusariumsubglutinans). A 1 to 9 visual rating indicating the resistance toFusarium ear rot. A higher score indicates a higher resistance.

GDU=GROWING DEGREE UNITS. Using the Barger Heat Unit Theory, whichassumes that maize growth occurs in the temperature range 50 degreesF.−86 degrees F. and that temperatures outside this range slow downgrowth; the maximum daily heat unit accumulation is 36 and the minimumdaily heat unit accumulation is 0. The seasonal accumulation of GDU is amajor factor in determining maturity zones.

GDUSHD=GDU TO SHED. The number of growing degree units (GDUs) or heatunits required for an inbred line or hybrid to have approximately 50percent of the plants shedding pollen and is measured from the time ofplanting. Growing degree units are calculated by the Barger Method,where the heat units for a 24-hour period are:

${GDU} = {\frac{\left( {{Max}.\mspace{14mu}{temp}.{+ {{M{in}}.\mspace{14mu}{temp}.}}} \right)}{2} - 50}$

The highest maximum temperature used is 86 degrees F. and the lowestminimum temperature used is 50 degrees F. For each inbred or hybrid ittakes a certain number of GDUs to reach various stages of plantdevelopment.

GDUSLK=GDU TO SILK. The number of growing degree units required for aninbred line or hybrid to have approximately 50 percent of the plantswith silk emergence from time of planting. Growing degree units arecalculated by the Barger Method as given in GDU SHD definition.

GENOTYPE. Refers to the genetic constitution of a cell or organism.

GIBERS=GIBBERELLA EAR ROT (PINK MOLD) (Gibberella zeae). A 1 to 9 visualrating indicating the resistance to Gibberella Ear Rot. A higher scoreindicates a higher resistance.

GIBROT=GIBBERELLA STALK ROT SCORE. Score of stalk rot severity due toGibberella (Gibberella zeae). Expressed as a 1 to 9 score with 9 beinghighly resistant.

GLFSPT=GRAY LEAF SPOT (Cercospora zeae-maydis). A 1 to 9 visual ratingindicating the resistance to Gray Leaf Spot. A higher score indicates ahigher resistance.

GOSWLT=GOSS' WILT (Corynebacterium nebraskense). A 1 to 9 visual ratingindicating the resistance to Goss' Wilt. A higher score indicates ahigher resistance.

GRNAPP=GRAIN APPEARANCE. This is a 1 to 9 rating for the generalappearance of the shelled grain as it is harvested based on such factorsas the color of harvested grain, any mold on the grain, and any crackedgrain. High scores indicate good grain visual quality.

HAPLOID PLANT PART. Refers to a plant part or cell that has the samehaploid genotype as PHACE.

HCBLT=HELMINTHOSPORIUM CARBONUM LEAF BLIGHT (Helminthosporium carbonum).A 1 to 9 visual rating indicating the resistance to Helminthosporiuminfection. A higher score indicates a higher resistance.

HD SMT=HEAD SMUT (Sphacelotheca reiliana). This score indicates thepercentage of plants not infected.

HSKCVR=HUSK COVER. A 1 to 9 score based on performance relative to keychecks, with a score of 1 indicating very short husks, tip of ear andkernels showing; 5 is intermediate coverage of the ear under mostconditions, sometimes with thin husk; and a 9 has husks extending andclosed beyond the tip of the ear. Scoring can best be done nearphysiological maturity stage or any time during dry down untilharvested.

INC D/A=GROSS INCOME (DOLLARS PER ACRE). Relative income per acreassuming drying costs of two cents per point above 15.5 percent harvestmoisture and current market price per bushel.

INCOME/ACRE. Income advantage of hybrid to be patented over other hybridon per acre basis.

INC ADV=GROSS INCOME ADVANTAGE. GROSS INCOME advantage of variety #1over variety #2.

KSZDCD=KERNEL SIZE DISCARD. The percent of discard seed; calculated asthe sum of discarded tip kernels and extra large kernels.

LINKAGE. Refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent.

LINKAGE DISEQUILIBRIUM. Refers to a phenomenon wherein alleles tend toremain together in linkage groups when segregating from parents tooffspring, with a greater frequency than expected from their individualfrequencies.

L/POP=YIELD AT LOW DENSITY. Yield ability at relatively low plantdensities on a 1 to 9 relative system with a higher number indicatingthe hybrid responds well to low plant densities for yield relative toother hybrids. A 1, 5, and 9 would represent very poor, average, andvery good yield response, respectively, to low plant density.

LRTLDG=LATE ROOT LODGING. Late root lodging is the percentage of plantsthat do not root lodge after anthesis through harvest; plants that leanfrom the vertical axis at an approximately 30 degree angle or greaterwould be counted as root lodged.

LRTLPN=LATE ROOT LODGING. Late root lodging is an estimate of thepercentage of plants that do not root lodge after anthesis throughharvest; plants that lean from the vertical axis at an approximately 30degree angle or greater would be considered as root lodged.

LRTLSC=LATE ROOT LODGING SCORE. Score for severity of plants that leanfrom a vertical axis at an approximate 30 degree angle or greater whichtypically results from strong winds after flowering. Recorded prior toharvest when a root-lodging event has occurred. This lodging results inplants that are leaned or “lodged” over at the base of the plant and donot straighten or “goose-neck” back to a vertical position. Expressed asa 1 to 9 score with 9 being no lodging.

MDMCPX=MAIZE DWARF MOSAIC COMPLEX (MDMV=Maize Dwarf Mosaic Virus andMCDV=Maize Chlorotic Dwarf Virus). A 1 to 9 visual rating indicating theresistance to Maize Dwarf Mosaic Complex. A higher score indicates ahigher resistance.

MST=HARVEST MOISTURE. The moisture is the actual percentage moisture ofthe grain at harvest.

MSTADV=MOISTURE ADVANTAGE. The moisture advantage of variety #1 overvariety #2 as calculated by: MOISTURE of variety #2-MOISTURE of variety#1=MOISTURE ADVANTAGE of variety #1.

NLFBLT=NORTHERN LEAF BLIGHT (Helminthosporium turcicum or Exserohilumturcicum). A 1 to 9 visual rating indicating the resistance to NorthernLeaf Blight. A higher score indicates a higher resistance.

OILT=GRAIN OIL. Absolute value of oil content of the kernel as predictedby Near-Infrared Transmittance and expressed as a percent of dry matter.

PEDIGREE DISTANCE. Relationship among generations based on theirancestral links as evidenced in pedigrees. May be measured by thedistance of the pedigree from a given starting point in the ancestry.

PERCENT IDENTITY. Percent identity as used herein refers to thecomparison of the homozygous alleles of two inbred lines. Each inbredplant will have the same allele (and therefore be homozygous) at almostall of their loci. Percent identity is determined by comparing astatistically significant number of the homozygous alleles of two inbredlines. For example, a percent identity of 90% between inbred PHACE andother inbred line means that the two inbred lines have the same alleleat 90% of their loci.

PERCENT SIMILARITY. Percent similarity as used herein refers to thecomparison of the homozygous alleles of an inbred line with anotherplant. The homozygous alleles of PHACE are compared with the alleles ofa non-inbred plant, such as a hybrid, and if the allele of the inbredmatches at least one of the alleles from the hybrid then they are scoredas similar. Percent similarity is determined by comparing astatistically significant number of loci and recording the number ofloci with similar alleles as a percentage. For example, a percentsimilarity of 90% between inbred PHACE and a hybrid maize plant meansthat the inbred line matches at least one of the hybrid alleles at 90%of the loci. In the case of a hybrid produced from PHACE as the male orfemale parent, such hybrid will comprise two sets of alleles, one set ofwhich will comprise the same alleles as the homozygous alleles of inbredline PHACE.

PLANT. As used herein, the term “plant” includes reference to animmature or mature whole plant, including a plant that has beendetasseled or from which seed or grain has been removed. Seed or embryothat will produce the plant is also considered to be the plant.

PLANT PARTS. As used herein, the term “plant parts” includes leaves,stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs,husks, stalks, root tips, anthers, pericarp, silk, tissue, cells and thelike.

PLTHT=PLANT HEIGHT. This is a measure of the height of the plant fromthe ground to the tip of the tassel in centimeters.

POLSC=POLLEN SCORE. A 1 to 9 visual rating indicating the amount ofpollen shed. The higher the score the more pollen shed.

POLWT=POLLEN WEIGHT. This is calculated by dry weight of tasselscollected as shedding commences minus dry weight from similar tasselsharvested after shedding is complete.

POP K/A=PLANT POPULATIONS. Measured as 1000s per acre.

POP ADV=PLANT POPULATION ADVANTAGE. The plant population advantage ofvariety #1 over variety #2 as calculated by PLANT POPULATION of variety#2-PLANT POPULATION of variety #1=PLANT POPULATION ADVANTAGE of variety#1.

PRM=PREDICTED RELATIVE MATURITY. This trait, predicted relativematurity, is based on the harvest moisture of the grain. The relativematurity rating is based on a known set of checks and utilizes standardlinear regression analyses and is also referred to as the ComparativeRelative Maturity Rating System that is similar to the MinnesotaRelative Maturity Rating System.

PRMSHD=A relative measure of the growing degree units (GDU) required toreach 50% pollen shed. Relative values are predicted values from thelinear regression of observed GDU's on relative maturity of commercialchecks.

PROT=GRAIN PROTEIN. Absolute value of protein content of the kernel aspredicted by Near-Infrared Transmittance and expressed as a percent ofdry matter.

RTLDG=ROOT LODGING. Root lodging is the percentage of plants that do notroot lodge; plants that lean from the vertical axis at an approximately30 degree angle or greater would be counted as root lodged.

RTLADV=ROOT LODGING ADVANTAGE. The root lodging advantage of variety #1over variety #2.

SCTGRN=SCATTER GRAIN. A 1 to 9 visual rating indicating the amount ofscatter grain (lack of pollination or kernel abortion) on the ear. Thehigher the score the less scatter grain.

SDGVGR=SEEDLING VIGOR. This is the visual rating (1 to 9) of the amountof vegetative growth after emergence at the seedling stage(approximately five leaves). A higher score indicates better vigor.

SEL IND=SELECTION INDEX. The selection index gives a single measure ofthe hybrid's worth based on information for up to five traits. A maizebreeder may utilize his or her own set of traits for the selectionindex. One of the traits that is almost always included is yield. Theselection index data presented in the tables represent the mean valueaveraged across testing stations.

SELF POLLINATION. A plant is self-pollinated if pollen from one floweris transferred to the same or another flower of the same plant.

SIB POLLINATION. A plant is sib-pollinated when individuals within thesame family or line are used for pollination.

SLFBLT=SOUTHERN LEAF BLIGHT (Helminthosporium maydis or Bipolarismaydis). A 1 to 9 visual rating indicating the resistance to SouthernLeaf Blight. A higher score indicates a higher resistance.

SOURST=SOUTHERN RUST (Puccinia polysora). A 1 to 9 visual ratingindicating the resistance to Southern Rust. A higher score indicates ahigher resistance.

STAGRN=STAY GREEN. Stay green is the measure of plant health near thetime of black layer formation (physiological maturity). A high scoreindicates better late-season plant health.

STDADV=STALK STANDING ADVANTAGE. The advantage of variety #1 overvariety #2 for the trait STK CNT.

STKCNT=NUMBER OF PLANTS. This is the final stand or number of plants perplot.

STKLDG=STALK LODGING REGULAR. This is the percentage of plants that didnot stalk lodge (stalk breakage) at regular harvest (when grain moistureis between about 20 and 30%) as measured by either natural lodging orpushing the stalks and determining the percentage of plants that breakbelow the ear.

STKLDL=LATE STALK LODGING. This is the percentage of plants that did notstalk lodge (stalk breakage) at or around late season harvest (whengrain moisture is between about 15 and 18%) as measured by eithernatural lodging or pushing the stalks and determining the percentage ofplants that break below the ear.

STKLDS=STALK LODGING SCORE. A plant is considered as stalk lodged if thestalk is broken or crimped between the ear and the ground. This can becaused by any or a combination of the following: strong winds late inthe season, disease pressure within the stalks, ECB damage orgenetically weak stalks. This trait should be taken just prior to or atharvest. Expressed on a 1 to 9 scale with 9 being no lodging.

STLLPN=LATE STALK LODGING. This is the percent of plants that did notstalk lodge (stalk breakage or crimping) at or around late seasonharvest (when grain moisture is below 20%) as measured by either naturallodging or pushing the stalks and determining the percentage of plantsthat break or crimp below the ear.

STLPCN=STALK LODGING REGULAR. This is an estimate of the percentage ofplants that did not stalk lodge (stalk breakage) at regular harvest(when grain moisture is between about 20 and 30%) as measured by eithernatural lodging or pushing the stalks and determining the percentage ofplants that break below the ear.

STRT=GRAIN STARCH. Absolute value of starch content of the kernel aspredicted by Near-Infrared Transmittance and expressed as a percent ofdry matter.

STWWLT=Stewart's Wilt (Erwinia stewartii). A 1 to 9 visual ratingindicating the resistance to Stewart's Wilt. A higher score indicates ahigher resistance.

TASBLS=TASSEL BLAST. A 1 to 9 visual rating was used to measure thedegree of blasting (necrosis due to heat stress) of the tassel at thetime of flowering. A 1 would indicate a very high level of blasting attime of flowering, while a 9 would have no tassel blasting.

TASSZ=TASSEL SIZE. A 1 to 9 visual rating was used to indicate therelative size of the tassel. The higher the rating the larger thetassel.

TAS WT=TASSEL WEIGHT. This is the average weight of a tassel (grams)just prior to pollen shed.

TEXEAR=EAR TEXTURE. A 1 to 9 visual rating was used to indicate therelative hardness (smoothness of crown) of mature grain. A 1 would bevery soft (extreme dent) while a 9 would be very hard (flinty or verysmooth crown).

TILLER=TILLERS. A count of the number of tillers per plot that couldpossibly shed pollen was taken. Data are given as a percentage oftillers: number of tillers per plot divided by number of plants perplot.

TSTWT=TEST WEIGHT (UNADJUSTED). The measure of the weight of the grainin pounds for a given volume (bushel).

TSWADV=TEST WEIGHT ADVANTAGE. The test weight advantage of variety #1over variety #2.

WIN M % =PERCENT MOISTURE WINS.

WIN Y % =PERCENT YIELD WINS.

YIELD BU/A=YIELD (BUSHELS/ACRE). Yield of the grain at harvest inbushels per acre adjusted to 15% moisture.

YLDADV=YIELD ADVANTAGE. The yield advantage of variety #1 over variety#2 as calculated by: YIELD of variety #1−YIELD variety #2=yieldadvantage of variety #1.

YLDSC=YIELD SCORE. A 1 to 9 visual rating was used to give a relativerating for yield based on plot ear piles. The higher the rating thegreater visual yield appearance.

Definitions for Area of Adaptability

When referring to area of adaptability, such term is used to describethe location with the environmental conditions that would be well suitedfor this maize line. Area of adaptability is based on a number offactors, for example: days to maturity, insect resistance, diseaseresistance, and drought resistance. Area of adaptability does notindicate that the maize line will grow in every location within the areaof adaptability or that it will not grow outside the area.

-   Central Corn Belt: Iowa, Illinois, Indiana-   Drylands: non-irrigated areas of North Dakota, South Dakota,    Nebraska, Kansas, Colorado and Oklahoma-   Eastern U.S.: Ohio, Pennsylvania, Delaware, Maryland, Virginia, and    West Virginia-   North central U.S.: Minnesota and Wisconsin-   Northeast: Michigan, New York, Vermont, and Ontario and Quebec    Canada-   Northwest U.S.: North Dakota, South Dakota, Wyoming, Washington,    Oregon, Montana, Utah, and Idaho-   South central U.S.: Missouri, Tennessee, Kentucky, Arkansas-   Southeast U.S.: North Carolina, South Carolina, Georgia, Florida,    Alabama, Mississippi, and Louisiana-   Southwest U.S.: Texas, Oklahoma, New Mexico, Arizona-   Western U.S.: Nebraska, Kansas, Colorado, and California-   Maritime Europe: Northern France, Germany, Belgium, Netherlands and    Austria

DETAILED DESCRIPTION OF THE INVENTION AND FURTHER EMBODIMENTS

All tables discussed in the Detailed Description of the Invention andFurther Embodiments section can found at the end of the section.

Morphological and Physiological Characteristics of PHACE

Inbred maize line PHACE is a yellow, dent maize inbred with some flintcharacteristics that may be used as either a male or female in theproduction of the first generation F1 maize hybrids. Inbred maize linePHACE is best adapted to the United States in the Central Corn Belt,Eastern, Southcentral, Western, U.S. Drylands and Southeast, and can beused to produce hybrids with approximately 113 maturity based on theComparative Relative Maturity Rating System for harvest moisture ofgrain. Inbred maize line PHACE demonstrates moderate tolerance toNorthern and Southern Leaf Blight, Anthracnose, and Diploidia. It hashigh extractable starch. In hybrid combination, inbred PHACEdemonstrates excellent yield over a wide area of adaptation. It hasadequate tolerance to stalk and root lodging and good tolerance tobrittle snap.

The inbred has shown uniformity and stability within the limits ofenvironmental influence for all the traits as described in the VarietyDescription Information (Table 1, found at the end of the section). Theinbred has been self-pollinated and ear-rowed a sufficient number ofgenerations with careful attention paid to uniformity of plant type toensure the homozygosity and phenotypic stability necessary for use incommercial hybrid seed production. The line has been increased both byhand and in isolated fields with continued observation for uniformity.No variant traits have been observed or are expected in PHACE.

Inbred maize line PHACE, being substantially homozygous, can bereproduced by planting seeds of the line, growing the resulting maizeplants under self-pollinating or sib-pollinating conditions withadequate isolation, and harvesting the resulting seed using techniquesfamiliar to the agricultural arts.

Genotypic Characteristics of PHACE

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile, which can identify plants of the same variety ora related variety, or be used to determine or validate a pedigree. TheSSR profile of Inbred PHACE can be found in Table 2 at the end of thissection.

As a result of inbreeding, PHACE is substantially homozygous. Thishomozygosity has been characterized at the loci shown in the markerprofile provided herein. An F1 hybrid made with PHACE would comprise themarker profile of PHACE shown herein. This is because an F1 hybrid isthe sum of its inbred parents, e.g., if one inbred parent is homozygousfor allele x at a particular locus, and the other inbred parent ishomozygous for allele y at that locus, the F1 hybrid will be x.y(heterozygous) at that locus. The profile can therefore be used toidentify hybrids comprising PHACE as a parent, since such hybrids willcomprise two sets of alleles, one set of which will be from PHACE. Thedetermination of the male set of alleles and the female set of allelesmay be made by profiling the hybrid and the pericarp of the hybrid seed,which is composed of maternal parent cells. The paternal parent profileis obtained by subtracting the pericarp profile from the hybrid profile.Subsequent generations of progeny produced by selection and breeding areexpected to be of genotype x (homozygous), y (homozygous), or x.y(heterozygous) for these locus positions. When the F1 plant is used toproduce an inbred, the resulting inbred should be either x or y for thatallele. In that regard, a unique allele or combination of alleles uniqueto that inbred can be used to identify progeny plants that retain thoseunique alleles or combinations of alleles.

Therefore, in accordance with the above, an embodiment of this inventionis a PHACE progeny maize plant or plant part that is a first generationhybrid maize plant comprising two sets of alleles, wherein one set ofthe alleles is the same as PHACE at all of the SSR loci listed in Table2. A maize cell wherein one set of the alleles is the same as PHACE atall of the SSR loci listed in Table 2 is also an embodiment of theinvention. This maize cell may be a part of a hybrid seed produced bycrossing PHACE with another inbred maize plant.

Genetic marker profiles can be obtained by techniques such asRestriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs) which are also referred to asMicrosatellites, and Single Nucleotide Polymorphisms (SNPs). Forexample, see Berry, Don, et al., “Assessing Probability of AncestryUsing Simple Sequence Repeat Profiles: Applications to Maize Hybrids andInbreds”, Genetics, 2002, 161:813–824, and Berry, Don, et al.,“Assessing Probability of Ancestry Using Simple Sequence RepeatProfiles: Applications to Maize Inbred Lines and Soybean Varieties”,Genetics, 2003, 165: 331–342, which are incorporated by referenceherein.

Particular markers used for these purposes are not limited to the set ofmarkers disclosed herein, but may include any type of marker and markerprofile which provides a means of distinguishing varieties. In additionto being used for identification of Inbred Line PHACE, a hybrid producedthrough the use of PHACE, and the identification or verification ofpedigree for progeny plants produced through the use of PHACE, thegenetic marker profile is also useful in further breeding and indeveloping an introgressed trait conversion of PHACE.

Means of performing genetic marker profiles using SSR polymorphisms arewell known in the art. SSRs are genetic markers based on polymorphismsin repeated nucleotide sequences, such as microsatellites. A markersystem based on SSRs can be highly informative in linkage analysisrelative to other marker systems in that multiple alleles may bepresent. Another advantage of this type of marker is that, through useof flanking primers, detection of SSRs can be achieved, for example, bythe polymerase chain reaction (PCR), thereby eliminating the need forlabor-intensive Southern hybridization. The PCR detection is done by useof two oligonucleotide primers flanking the polymorphic segment ofrepetitive DNA. Repeated cycles of heat denaturation of the DNA followedby annealing of the primers to their complementary sequences at lowtemperatures, and extension of the annealed primers with DNA polymerase,comprise the major part of the methodology.

Following amplification, markers can be scored by electrophoresis of theamplification products. Scoring of marker genotype is based on the sizeof the amplified fragment, which may be measured by the base pair weightor molecular weight of the fragment. While variation in the primer usedor in laboratory procedures can affect the reported fragment size,relative values should remain constant regardless of the specific primeror laboratory used. When comparing lines it is preferable if all SSRprofiles are performed in the same lab. The SSR analyses reported hereinwere conducted in-house at Pioneer Hi-Bred. An SSR service is availableto the public on a contractual basis by DNA Landmarks inSaint-Jean-sur-Richelieu, Quebec, Canada (formerly Paragen, CeleraAgGen, Perkin-Elmer AgGen, Linkage Genetics and NPI).

Primers used for the SSRs reported herein are publicly available and maybe found in the Maize GDB on the World Wide Web at maizegdb.org(sponsored by the USDA Agricultural Research Service), in Sharopova etal. (Plant Mol. Biol. 48(5–6):463–481), Lee et al (Plant Mol. Biol.48(5–6); 453–461), or may be constructed from sequences if reportedherein. Primers may be constructed from publicly available sequenceinformation. Some marker information may also be available from DNALandmarks.

Map information is provided by bin number as reported in the Maize GDBfor the IBM 2 and/or IBM 2 Neighbors maps. The bin number digits to theleft of decimal point represent the chromosome on which such marker islocated, and the digits to the right of the decimal represent thelocation on such chromosome. A bin number.xx designation indicates thatthe bin location on that chromosome is not known. Map positions are alsoavailable on the Maize GDB for a variety of different mappingpopulations.

PHACE and its plant parts can be identified through molecular markerprofile. An inbred corn plant cell having the SSR genetic marker profileshown in Table 2 is an embodiment of the invention. Such cell may beeither diploid or haploid.

Also encompassed within the scope of the invention are plants and plantparts substantially benefiting from the use of PHACE in theirdevelopment, such as PHACE comprising a introgressed trait throughbackcross conversion or transformation, and which may be identified byhaving an SSR molecular marker profile with a high percent identity toPHACE, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity.Likewise, percent similarity at these percentages may be used toidentify hybrid and other non-inbred plants produced by the use ofPHACE.

An embodiment of this invention is an inbred PHACE progeny maize plantor plant part comprising the same homozygous alleles as the plant orplant part of PHACE for at least 90% of the SSR loci listed in Table 2.A plant cell comprising the same homozygous alleles as a plant cell ofPHACE for at least 90% of the SSR loci listed in Table 2 is also anembodiment of this invention. In these specific embodiments, 90% mayalso be replaced by any integer or partial integer percent of 80% orgreater as listed above. One means of producing such a progeny plant,plant part or cell is through the backcrossing and/or transformationmethods described herein.

Similarly, an embodiment of this invention is a PHACE progeny maizeplant or plant part comprising at least one allele per locus that is thesame allele as the plant or plant part of PHACE for at least 90% of theSSR loci listed in Table 2. This progeny plant may be a hybrid. Aprogeny or hybrid plant cell wherein at least one allele per locus thatis the same allele as the plant cell PHACE for at least 90% of the SSRloci listed in Table 2 is also a specific embodiment of this invention.In these specific embodiments, 90% may also be replaced by any integerpercent listed above. One means of producing such a progeny or hybridplant, plant part or cell is through the backcrossing and/ortransformation methods described herein.

In addition, the SSR profile of PHACE also can be used to identifyessentially derived varieties and other progeny lines developed from theuse of PHACE, as well as cells and other plant parts thereof. Progenyplants and plant parts produced using PHACE may be identified by havinga molecular marker profile of at least 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% geneticcontribution from inbred line PHACE, as measured by either percentidentity or percent similarity.

Comparing PHACE To Other Inbreds

A breeder uses various methods to help determine which plants should beselected from segregating populations and ultimately which inbred lineswill be used to develop hybrids for commercialization. In addition toknowledge of the germplasm and plant genetics, a part of the selectionprocess is dependent on experimental design coupled with the use ofstatistical analysis. Experimental design and statistical analysis areused to help determine which plants, which family of plants, and finallywhich inbred lines and hybrid combinations are significantly better ordifferent for one or more traits of interest. Experimental designmethods are used to assess error so that differences between two inbredlines or two hybrid lines can be more accurately evaluated. Statisticalanalysis includes the calculation of mean values, determination of thestatistical significance of the sources of variation, and thecalculation of the appropriate variance components. Either a five or aone percent significance level is customarily used to determine whethera difference that occurs for a given trait is real or due to theenvironment or experimental error. One of ordinary skill in the art ofplant breeding would know how to evaluate the traits of two plantvarieties to determine if there is no significant difference between thetwo traits expressed by those varieties. For example, see Fehr, Walt,Principles of Cultivar Development, p. 261–286 (1987) which isincorporated herein by reference. Mean trait values may be used todetermine whether trait differences are significant. Trait values shouldpreferably be measured on plants grown under the same environmentalconditions, and environmental conditions should be appropriate for thetraits or traits being evaluated. Sufficient selection pressure shouldbe present for optimum measurement of traits of interest such asherbicide, insect or disease resistance. Similarly, an introgressedtrait conversion of PHACE for resistance, such as herbicide resistance,should not be compared to PHACE in the presence of the herbicide whencomparing non-resistance related traits such as plant height and yield.

In Table 3, data from traits and characteristics of inbred maize linePHACE per se are given and compared to other maize inbred lines andhybrids. The following are the results of these comparisons:

The results in Table 3 A compare inbred PHACE to inbred PH6 ME. Theresults show inbred PHACE has significantly morenumber of tillers perplot and taller plant height compared to PH6ME.

The results in Table 3B compare inbred PHACE to inbred PH24E. Theresults show inbred PHACE differs significantly over multiple traitsincluding higher yield and number of tillers when compared to inbredPH24E.

Development of Maize Hybrids Using PHACE

A single cross maize hybrid results from the cross of two inbred lines,each of which has a genotype that complements the genotype of the other.The hybrid progeny of the first generation is designated F1. In thedevelopment of commercial hybrids in a maize plant breeding program,only the F1 hybrid plants are sought. F1 hybrids are more vigorous thantheir inbred parents. This hybrid vigor, or heterosis, can be manifestedin many polygenic traits, including increased vegetative growth andincreased yield.

PHACE may be used to produce hybrid maize. One such embodiment is themethod of crossing inbred maize line PHACE with another maize plant,such as a different maize inbred line, to form a first generation F1hybrid seed. The first generation F1 hybrid seed, plant and plant partproduced by this method is an embodiment of the invention. The firstgeneration F1 seed, plant and plant part will comprise an essentiallycomplete set of the alleles of inbred line PHACE. One of ordinary skillin the art can utilize either breeder books or molecular methods toidentify a particular F1 hybrid plant produced using inbred line PHACE.Further, one of ordinary skill in the art may also produce F1 hybridswith transgenic, male sterile and/or backcross conversions of inbredline PHACE.

The development of a maize hybrid in a maize plant breeding programinvolves three steps: (1) the selection of plants from various germplasmpools for initial breeding crosses; (2) the selfing of the selectedplants from the breeding crosses for several generations to produce aseries of inbred lines, such as PHACE, which, although different fromeach other, breed true and are highly uniform; and (3) crossing theselected inbred lines with different inbred lines to produce thehybrids. During the inbreeding process in maize, the vigor of the linesdecreases, and so one would not be likely to use PHACE directly toproduce grain. However, vigor is restored when PHACE is crossed to adifferent inbred line to produce a commercial F1 hybrid. An importantconsequence of the homozygosity and homogeneity of the inbred line isthat the hybrid between a defined pair of inbreds may be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained.

PHACE may be used to produce a single cross hybrid, a three-way hybridor a double cross hybrid. A single cross hybrid is produced when twoinbred lines are crossed to produce the F1 progeny. A double crosshybrid is produced from four inbred lines crossed in pairs (A×B and C×D)and then the two F1 hybrids are crossed again (A×B)×(C×D). A three-waycross hybrid is produced from three inbred lines where two of the inbredlines are crossed (A×B) and then the resulting F1 hybrid is crossed withthe third inbred (A×B)×C. In each case, pericarp tissue from the femaleparent will be a part of and protect the hybrid seed.

Combining Ability of PHACE

Combining ability of a line, as well as the performance of the line perse, is a factor in the selection of improved maize inbreds. Combiningability refers to a line's contribution as a parent when crossed withother lines to form hybrids. The hybrids formed for the purpose ofselecting superior lines may be referred to as test crosses, and includecomparisons to other hybrid varieties grown in the same environment(same cross, location and time of planting). One way of measuringcombining ability is by using values based in part on the overall meanof a number of test crosses weighted by number of experiment andlocation combinations in which the hybrid combinations occurs. The meanmay be adjusted to remove environmental effects and known geneticrelationships among the lines.

General combining ability provides an overall score for the inbred overa large number of test crosses. Specific combining ability providesinformation on hybrid combinations formed by PHACE and a specific inbredparent. A line such as PHACE which exhibits good general combiningability may be used in a large number of hybrid combinations.

A general combining ability report for inbred PHACE is provided in Table4. This data represents the overall mean value for these traits overhundreds of test crosses. Table 4 demonstrates that inbred PHACE showsgood general combining ability for hybrid production.

Hybrid Comparisons

These hybrid comparisons represent specific hybrid crosses with PHACEand a comparison of these specific hybrids with other hybrids withfavorable characteristics. These comparisons illustrate the goodspecific combining ability of PHACE.

The results in Table 5A compare a specific hybrid for which inbred PHACEis a parent and a second hybrid, 33M54. The results show that the hybridcontaining inbred PHACE produced significantly different results overmultiple traits including moisture, number of growing degree unitsrequired to achieve 50% pollen shed and yield.

The results in Table 5B compare a specific hybrid for which inbred PHACEis a parent and a second hybrid, 34B97. The results show that the hybridcontaining inbred PHACE produced numerically different results overmultiple traits including plant height and ear height.

Introgression of a New Locus or Trait into PHACE.

PHACE represents a new base genetic line into which a new locus or traitmay be introgressed. Direct transformation and backcrossing representtwo important methods that can be used to accomplish such anintrogression. The term backcross conversion and single locus conversionare used interchangeably to designate the product of a backcrossingprogram.

Backcross Conversions of PHACE

A backcross conversion of PHACE occurs when DNA sequences are introducedthrough backcrossing (Hallauer et al, 1988), with PHACE utilized as therecurrent parent. Both naturally occurring and transgenic DNA sequencesmay be introduced through backcrossing techniques. A backcrossconversion may produce a plant with a trait or locus conversion in atleast one or more backcrosses, including at least 2 crosses, at least 3crosses, at least 4 crosses, at least 5 crosses and the like. Molecularmarker assisted breeding or selection may be utilized to reduce thenumber of backcrosses necessary to achieve the backcross conversion. Forexample, see Openshaw, S. J. et al., Marker-assisted Selection inBackcross Breeding. In: Proceedings Symposium of the Analysis ofMolecular Data, August 1994, Crop Science Society of America, Corvallis,Oreg., where it is demonstrated that a backcross conversion can be madein as few as two backcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes as vs.unlinked genes), the level of expression of the trait, the type ofinheritance (cytoplasmic or nuclear) and the types of parents includedin the cross. It is understood by those of ordinary skill in the artthat for single gene traits that are relatively easy to classify, thebackcross method is effective and relatively easy to manage. (SeeHallauer et al. in Corn and Corn Improvement, Sprague and Dudley, ThirdEd. 1998). Desired traits that may be transferred through backcrossconversion include, but are not limited to, waxy starch, sterility(nuclear and cytoplasmic), fertility restoration, grain color (white),nutritional enhancements, drought tolerance, nitrogen utilization,altered fatty acid profile, increased digestibility, low phytate,industrial enhancements, disease resistance (bacterial, fungal orviral), insect resistance, herbicide resistance and yield enhancements.In addition, an introgression site itself, such as an FRT site, Lox siteor other site specific integration site, may be inserted by backcrossingand utilized for direct insertion of one or more genes of interest intoa specific plant variety. In some embodiments of the invention, thenumber of loci that may be backcrossed into PHACE is at least 1, 2, 3,4, or 5 and/or no more than 6, 5, 4, 3, or 2. A single loci may containseveral transgenes, such as a transgene for disease resistance that, inthe same expression vector, also contains a transgene for herbicideresistance. The gene for herbicide resistance may be used as aselectable marker and/or as a phenotypic trait. A single locusconversion of site specific integration system allows for theintegration of multiple genes at the converted loci.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele, such as the waxy starchcharacteristic, requires growing and selfing the first backcrossgeneration to determine which plants carry the recessive alleles.Recessive traits may require additional progeny testing in successivebackcross generations to determine the presence of the locus ofinterest. The last backcross generation is usually selfed to give purebreeding progeny for the gene(s) being transferred, although a backcrossconversion with a stably introgressed trait may also be maintained byfurther backcrossing to the recurrent parent with selection for theconverted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype of the recurrent parent. While occasionally additionalpolynucleotide sequences or genes may be transferred along with thebackcross conversion, the backcross conversion line “fits into the samehybrid combination as the recurrent parent inbred line and contributesthe effect of the additional gene added through the backcross.” Poehlmanet al. (1995, page 334). It has been proposed that in general thereshould be at least four backcrosses when it is important that therecovered lines be essentially identical to the recurrent parent exceptfor the characteristic being transferred (Fehr 1987, Principles ofCultivar Development). However, as noted above, the number ofbackcrosses necessary can be reduced with the use of molecular markers.Other factors, such as a genetically similar donor parent, may alsoreduce the number of backcrosses necessary.

One process for adding or modifying a trait or locus in maize inbredline PHACE comprises crossing PHACE plants grown from PHACE seed withplants of another maize line that comprise the desired trait or locus,selecting F1 progeny plants that comprise the desired trait or locus toproduce selected F1 progeny plants, crossing the selected progeny plantswith the PHACE plants to produce backcross progeny plants, selecting forbackcross progeny plants that have the desired trait or locus and themorphological characteristics of maize inbred line PHACE to produceselected backcross progeny plants; and backcrossing to PHACE three ormore times in succession to produce selected fourth or higher backcrossprogeny plants that comprise said trait or locus. The modified PHACE maybe further characterized as having the physiological and morphologicalcharacteristics of maize inbred line PHACE listed in Table 1 asdetermined at the 5% significance level when grown in the sameenvironmental conditions and/or may be characterized by percentsimilarity or identity to PHACE as determined by SSR markers. The abovemethod may be utilized with fewer backcrosses in appropriate situations,such as when the donor parent is highly related or markers are used inthe selection step. Desired traits that may be used include thosenucleic acids known in the art, some of which are listed herein, thatwill affect traits through nucleic acid expression or inhibition.Desired loci include the introgression of FRT, Lox and other sites forsite specific integration, which may also affect a desired trait if afunctional nucleic acid is inserted at the integration site.

In addition, the above process and other similar processes describedherein may be used to produce F1 hybrid maize seed by adding a step atthe end of the process that comprises crossing PHACE with theintrogressed trait or locus with a different maize plant and harvestingthe resultant F1 hybrid maize seed.

Male Sterility and Hybrid Seed Production

Hybrid seed production requires elimination or inactivation of pollenproduced by the female inbred parent. Incomplete removal or inactivationof the pollen provides the potential for self-pollination. A reliablemethod of controlling male fertility in plants offers the opportunityfor improved seed production.

PHACE can be produced in a male-sterile form. There are several ways inwhich a maize plant can be manipulated so that it is male sterile. Theseinclude use of manual or mechanical emasculation (or detasseling), useof one or more genetic factors that confer male sterility, includingcytoplasmic genetic and/or nuclear genetic male sterility, use ofgametocides and the like. A male sterile inbred designated PHACE mayinclude one or more genetic factors, which result in cytoplasmic geneticand/or nuclear genetic male sterility. All of such embodiments arewithin the scope of the present claims. The male sterility may be eitherpartial or complete male sterility.

Hybrid maize seed is often produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twomaize inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Provided that there issufficient isolation from sources of foreign maize pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

The laborious detasseling process can be avoided by using cytoplasmicmale-sterile (CMS) inbreds. Plants of a CMS inbred are male sterile as aresult of genetic factors in the cytoplasm, as opposed to the nucleus,and so nuclear linked genes are not transferred during backcrossing.Thus, this characteristic is inherited exclusively through the femaleparent in maize plants, since only the female provides cytoplasm to thefertilized seed. CMS plants are fertilized with pollen from anotherinbred that is not male-sterile. Pollen from the second inbred may ormay not contribute genes that make the hybrid plants male-fertile, andeither option may be preferred depending on the intended use of thehybrid. The same hybrid seed, a portion produced from detasseled fertilemaize and a portion produced using the CMS system, can be blended toinsure that adequate pollen loads are available for fertilization whenthe hybrid plants are grown. CMS systems have been successfully usedsince the 1950's, and the male sterility trait is routinely backcrossedinto inbred lines. See Wych, p. 585–586, 1998.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

These, and the other methods of conferring genetic male sterility in theart, each possess their own benefits and drawbacks. Some other methodsuse a variety of approaches such as delivering into the plant a geneencoding a cytotoxic substance associated with a male tissue specificpromoter or an antisense system in which a gene critical to fertility isidentified and an antisense to that gene is inserted in the plant (see:Fabinjanski, et al. EPO 89/3010153.8 publication No. 329,308 and PCTapplication PCT/CA90/00037 published as WO 90/08828).

Another system for controlling male sterility makes use of gametocides.Gametocides are not a genetic system, but rather a topical applicationof chemicals. These chemicals affect cells that are critical to malefertility. The application of these chemicals affects fertility in theplants only for the growing season in which the gametocide is applied(see Carlson, Glenn R., U.S. Pat. No. 4,936,904). Application of thegametocide, timing of the application and genotype specificity oftenlimit the usefulness of the approach and it is not appropriate in allsituations.

Incomplete control over male fertility may result in self-pollinatedseed being unintentionally harvested and packaged with hybrid seed. Thiswould typically be only female parent seed, because the male plant isgrown in rows that are typically destroyed prior to seed development.Once the seed from the hybrid bag is planted, it is possible to identifyand select these self-pollinated plants. These self-pollinated plantswill be genetically equivalent to one of the inbred lines used toproduce the hybrid. Though the possibility of inbred PHACE beingincluded in a hybrid seed bag exists, the occurrence is very low becausemuch care is taken by seed companies to avoid such inclusions. It isworth noting that hybrid seed is sold to growers for the production ofgrain or forage and not for breeding or seed production. Theseself-pollinated plants can be identified and selected by one skilled inthe art due to their less vigorous appearance for vegetative and/orreproductive characteristics, including shorter plant height, small earsize, ear and kernel shape, cob color, or other characteristics.

Identification of these self-pollinated lines can also be accomplishedthrough molecular marker analyses. See, “The Identification of FemaleSelfs in Hybrid Maize: A Comparison Using Electrophoresis andMorphology”, Smith, J. S. C. and Wych, R. D., Seed Science andTechnology 14, pp. 1–8 (1995), the disclosure of which is expresslyincorporated herein by reference. Through these technologies, thehomozygosity of the self pollinated line can be verified by analyzingallelic composition at various loci along the genome. Those methodsallow for rapid identification of the invention disclosed herein. Seealso, “Identification of Atypical Plants in Hybrid Maize Seed byPostcontrol and Electrophoresis” Sarca, V. et al., Probleme de GeneticaTeoritica si Aplicata Vol. 20 (1) p. 29–42.

An embodiment of this invention is a process for producing seed ofPHACE, comprising planting a collection of seed comprising seed of ahybrid, one of whose parents is inbred PHACE, said collection alsocomprising seed of said inbred, growing plants from said collection ofseed, identifying inbred parent plants, selecting said inbred parentplant; and controlling pollination to preserve the homozygosity of saidinbred parent plant.

Transformation

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions, such as encoding specific protein products. Scientists in thefield of plant biology developed a strong interest in engineering thegenome of plants to contain and express foreign genetic elements, oradditional, or modified versions of native or endogenous geneticelements in order to alter the traits of a plant in a specific manner.Any DNA sequences, whether from a different species or from the samespecies, that are inserted into the genome using transformation arereferred to herein collectively as “transgenes”. In some embodiments ofthe invention, a transformed variant of PHACE may contain at least onetransgene but could contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10and/or no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.Over the last fifteen to twenty years several methods for producingtransgenic plants have been developed, and the present invention alsorelates to transformed versions of the claimed inbred maize line PHACEas well as hybrid combinations thereof.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67–88 and Armstrong, “The First Decade of Maize Transformation: A Reviewand Future Perspective” (Maydica 44:101–109, 1999). In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber et al., “Vectors for Plant Transformation” in Methods inPlant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89–119.

The most prevalent types of plant transformation involve theconstruction of an expression vector. Such a vector comprises a DNAsequence that contains a gene under the control of or operatively linkedto a regulatory element, for example a promoter. The vector may containone or more genes and one or more regulatory elements.

A genetic trait which has been engineered into the genome of aparticular maize plant using transformation techniques, could be movedinto the genome of another line using traditional breeding techniquesthat are well known in the plant breeding arts. For example, abackcrossing approach is commonly used to move a transgene from atransformed maize plant to an elite inbred line, and the resultingprogeny would then comprise the transgene(s). Also, if an inbred linewas used for the transformation then the transgenic plants could becrossed to a different inbred in order to produce a transgenic hybridmaize plant.

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to genes;coding sequences; inducible, constitutive, and tissue specificpromoters; enhancing sequences; and signal and targeting sequences. Forexample, see the traits, genes and transformation methods listed in U.S.Pat. No. 6,118,055.

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants that areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114: 92–6(1981).

A genetic map can be generated, primarily via conventional RestrictionFragment Length Polymorphisms (RFLP), Polymerase Chain Reaction (PCR)analysis, Simple Sequence Repeats (SSR) and Single NucleotidePolymorphisms (SNP) that identifies the approximate chromosomal locationof the integrated DNA molecule. For exemplary methodologies in thisregard, see Glick and Thompson, METHODS IN PLANT MOLECULAR BIOLOGY ANDBIOTECHNOLOGY 269–284 (CRC Press, Boca Raton, 1993).

Wang et al. discuss “Large Scale Identification, Mapping and Genotypingof Single-Nucleotide Polymorphisms in the Human Genome”, Science,280:1077–1082, 1998, and similar capabilities are becoming increasinglyavailable for the corn genome. Map information concerning chromosomallocation is useful for proprietary protection of a subject transgenicplant. If unauthorized propagation is undertaken and crosses made withother germplasm, the map of the integration region can be compared tosimilar maps for suspect plants to determine if the latter have a commonparentage with the subject plant. Map comparisons would involvehybridizations, RFLP, PCR, SSR and sequencing, all of which areconventional techniques. SNPs may also be used alone or in combinationwith other techniques.

Likewise, by means of the present invention, plants can be geneticallyengineered to express various phenotypes of agronomic interest. Throughthe transformation of maize the expression of genes can be modulated toenhance disease resistance, insect resistance, herbicide resistance,agronomic, grain quality and other traits. Transformation can also beused to insert DNA sequences which control or help controlmale-sterility. DNA sequences native to maize as well as non-native DNAsequences can be transformed into maize and used to modulate levels ofnative or non-native proteins. Various promoters, targeting sequences,enhancing sequences, and other DNA sequences can be inserted into themaize genome for the purpose of modulating the expression of proteins.Reduction of the activity of specific genes (also known as genesilencing, or gene suppression) is desirable for several aspects ofgenetic engineering in plants.

Many techniques for gene silencing are well known to one of skill in theart, including but not limited to antisense technology (see, e.g.,Sheehy et al. (1988) PNAS USA 85:8805–8809; and U.S. Pat. Nos.5,107,065; 5,453,566; and 5,759,829); co-suppression (e.g., Taylor(1997) Plant Cell 9:1245; Jorgensen (1990) Trends Biotech.8(12):340–344; Flavell (1994) PNAS USA 91:3490–3496; Finnegan et al.(1994) Bio/Technology 12: 883–888; and Neuhuber et al. (1994) Mol. Gen.Genet. 244:230–241); RNA interference (Napoli et al. (1990) Plant Cell2:279–289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev. 13:139–141;Zamore et al. (2000) Cell 101:25–33; and Montgomery et al. (1998) PNASUSA 95:15502–15507), virus-induced gene silencing (Burton, et al. (2000)Plant Cell 12:691–705; and Baulcombe (1999) Curr. Op. Plant Bio.2:109–113); target-RNA-specific ribozymes (Haseloff et al. (1988) Nature334: 585–591); hairpin structures (Smith et al. (2000) Nature407:319–320; WO 99/53050; and WO 98/53083); ribozymes (Steinecke et al.((1992) EMBO J. 11:1525; and Perriman et al. ((1993) Antisense Res. Dev.3:253); oligonucleotide mediated targeted modification (e.g., WO03/076574 and WO 99/25853); Zn-finger targeted molecules (e.g., WO01/52620; WO 03/048345; and WO 00/42219); and other methods orcombinations of the above methods known to those of skill in the art.

Exemplary transgenes useful for genetic engineering include, but are notlimited to, those categorized below.

1. Transgenes that Confer Resistance to Pests or Disease and thatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266: 789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae). A plant resistant to a disease is one that ismore resistant to a pathogen as compared to the wild type plant.

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence ofa Bt delta-endotoxin gene. Moreover, DNA molecules encodingdelta-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), for example, under ATCC Accession Nos.40098, 67136, 31995 and 31998. Other examples of Bacillus thuringiensistransgenes being genetically engineered are given in the followingpatents and patent applications and hereby are incorporated by referencefor this purpose: U.S. Pat. Nos. 5,188,960; 5,689,052; 5,880,275; WO91/114778; WO 99/31248; WO 01/12731; WO 99/24581; WO 97/40162 and U.S.application Ser. Nos. 10/032,717; 10/414,637; and 10/606,320.

(C) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(D) An insect-specific peptide which, upon expression, disrupts thephysiology of the affected pest. For example, see the disclosures ofRegan, J. Biol. Chem. 269: 9 (1994) (expression cloning yields DNAcoding for insect diuretic hormone receptor), and Pratt et al., Biochem.Biophys. Res. Comm. 163: 1243 (1989) (an allostatin is identified inDiploptera puntata). See also U.S. Pat. No.5,266,317 to Tomalski et al.,who disclose genes encoding insect-specific toxins.

(E) An enzyme responsible for an hyperaccumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(F) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene.

(G) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104: 1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

(H) A hydrophobic moment peptide. See PCT application WO 95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference for this purpose.

(I) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-beta lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(J) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

(K) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(L) A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366: 469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(M) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo alpha-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-alpha-1,4-D-galacturonase. See Lamb etal., Bio/Technology 10: 1436 (1992). The cloning and characterization ofa gene which encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2: 367 (1992).

(N) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, S., Current Biology, 5(2)(1995).

(P) Antifungal genes (Cornelissen and Melchers, Pl. Physiol.101:709–712, (1993) and Parijs et al., Planta 183:258–264, (1991) andBushnell et al., Can. J. of Plant Path. 20(2):137–149 (1998).

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see U.S. Pat. No. 5,792,931.

(R) Cystatin and cysteine proteinase inhibitors.

(S) Defensin genes. See WO03000863.

(T) Genes conferring resistance to nematodes. See WO 03/033651 and Urwinet. al., Planta 204:472–479 (1998).

2. Transgenes that Confer Resistance to a Herbicide, for Example:

(A) A herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl. Genet. 80: 449(1990), respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659;5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107;5,928,937; and 5,378,824; and international publication WO 96/33270,which are incorporated herein by reference for this purpose.

(B) Glyphosate (resistance imparted by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin acetyl transferase (bar) genes), andpyridinoxy or phenoxy proprionic acids and cycloshexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. U.S. Pat. No. 5,627,061 to Barryet al. also describes genes encoding EPSPS enzymes. See also U.S. Pat.Nos. 6,248,876 B1; 6,040,497; 5,804,425; 5,633,435; 5,145,783;4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114 B1;6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re.36,449; RE 37,287 E; and 5,491,288; and international publications WO97/04103; WO 97/04114; WO 00/66746; WO 01/66704; WO 00/66747 and WO00/66748, which are incorporated herein by reference for this purpose.Glyphosate resistance is also imparted to plants that express a genethat encodes a glyphosate oxido-reductase enzyme as described more fullyin U.S. Pat. Nos. 5,776,760 and 5,463,175, which are incorporated hereinby reference for this purpose. In addition glyphosate resistance can beimparted to plants by the over expression of genes encoding glyphosateN-acetyltransferase. See, for example, U.S. application Ser. Nos.60/244,385; 60/377,175 and 60/377,719. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European patent application No. 0 333 033 to Kumadaet al. and U.S. Pat. No. 4,975,374 to Goodman et al. disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in European PatentNo. 0 242 246 and 0 242 236 to Leemans et al. De Greef et al.,Bio/Technology 7: 61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. See also, U.S. Pat. Nos. 5,969,213;5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477;5,646,024; 6,177,616 B1; and 5,879,903, which are incorporated herein byreference for this purpose. Exemplary genes conferring resistance tophenoxy proprionic acids and cycloshexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83: 435 (1992).

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

(D) Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants (see, e.g., Hattori et al. (1995)Mol Gen Genet 246:419). Other genes that confer tolerance to herbicidesinclude: a gene encoding a chimeric protein of rat cytochrome P4507A1and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et al. (1994)Plant PhysiolPlant Physiol 106:17), genes for glutathione reductase andsuperoxide dismutase (Aono et al. (1995) Plant Cell Physiol 36:1687, andgenes for various phosphotransferases (Datta et al. (1992) Plant MolBiol 20:619).

(E) Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1;and 5,767,373; and international publication WO 01/12825, which areincorporated herein by reference for this purpose.

3. Transgenes that Confer or Contribute to a Grain Trait, Such as:

(A) Modified fatty acid metabolism, for example, by

-   -   (1) Transforming a plant with an antisense gene of stearoyl-ACP        desaturase to increase stearic acid content of the plant. See        Knultzon et al., Proc. Natl. Acad. Sci. USA 89: 2624 (1992),    -   (2) Elevating oleic acid via FAD-2 gene modification and/or        decreasing linolenic acid via FAD-3 gene modification (see U.S.        Pat. Nos. 6,063,947; 6,323,392; and WO 93/11245),    -   (3) Altering conjugated linolenic or linoleic acid content, such        as in WO 01/12800,    -   (4) Modifying LEC1, AGP, Dek1, Superal1, thioredoxin, and/or a        gamma zein knock out or mutant such as cs27 or TUSC 27. For        example, see WO 02/42424, WO 98/22604, WO 03/011015, U.S. Pat.        No. 6,423,886 and Rivera-Madrid, R. et. al. Proc. Natl. Acad.        Sci. 92:5620–5624 (1995).

(B) Decreased phytate content, for example, by the

-   -   (1) Introduction of a phytase-encoding gene would enhance        breakdown of phytate, adding more free phosphate to the        transformed plant. For example, see Van Hartingsveldt et al.,        Gene 127: 87 (1993), for a disclosure of the nucleotide sequence        of an Aspergillus niger phytase gene.    -   (2) Introduction of a gene that reduces phytate content. In        maize, this, for example, could be accomplished, by cloning and        then re-introducing DNA associated with one or more of the        alleles, such as the LPA alleles, identified in maize mutants        characterized by low levels of phytic acid, such as in Raboy et        al., Maydica 35: 383 (1990) and/or by altering inositol kinase        activity as in WO 02/059324, US2003/0009011, WO 03/027243,        US2003/0079247 and WO 99/05298.

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170: 810(1988) (nucleotide sequence of Streptococcus mutans fructosyltransferasegene), Steinmetz et al, Mol. Gen. Genet. 200: 220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al,Bio/Technology 10: 292 (1992) (production of transgenic plants thatexpress Bacillus lichenifornis alpha-amylase), Elliot et al, PlantMolec. Biol. 21: 515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al, J. Biol. Chem. 268: 22480 (1993) (site-directedmutagenesis of barley alpha-amylase gene), and Fisher et al, PlantPhysiol. 102: 1045 (1993) (maize endosperm starch branching enzyme 11).The fatty acid modification genes mentioned above may also be used toeffect starch content and/or composition through the interrelationshipof the starch and oil pathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see WO 00/68393 involving themanipulation of antioxidant levels through alteration of a phytl prenyltransferase and WO 03/082899 through alteration of a homogentisategeranyl geranyl transferase.

(E) Improved digestibility and/or starch extraction through modificationof UDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref1, HCHL, C4H, such asin WO 99/10498.

4. Genes that Control Male-sterility

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT (WO 01/29237).

(B) Introduction of various stamen-specific promoters (WO 92/13956, WO92/13957).

(C) Introduction of the barnase and the barstar gene (Paul et al. PlantMol. Biol. 19:611–622, 1992).

5. Genes that create a site for site specific DNA integration. Thisincludes the introduction of FRT sites that may be used in the FLP/FRTsystem and/or Lox sites that may be used in the Cre/Loxp system. Forexample, see Lyznik, et al., Site-Specific Recombination for GeneticEngineering in Plants, Plant Cell Rep (2003) 21:925–932 and WO 99/25821,which are hereby incorporated by reference. Other systems that may beused include the Gin recombinase of phage Mu (Maeser et al., 1991), thePin recombinase of E. coli (Enomoto et al., 1983), and the R/RS systemof the pSR1 plasmid (Araki et al., 1992).6. Genes that affect growth characteristics, such as drought toleranceand nitrogen utilization. For example, see WO 00/73475 where water useefficiency is modulated through alteration of malate.Using PHACE to Develop Other Maize Inbreds

Inbred maize lines such as PHACE are typically developed for use in theproduction of hybrid maize lines. However, inbred lines such as PHACEalso provide a source of breeding material that may be used to developnew maize inbred lines. Plant breeding techniques known in the art andused in a maize plant breeding program include, but are not limited to,recurrent selection, mass selection, bulk selection, mass selection,backcrossing, pedigree breeding, open pollination breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection, making double haploids, and transformation. Oftencombinations of these techniques are used. The development of maizehybrids in a maize plant breeding program requires, in general, thedevelopment of homozygous inbred lines, the crossing of these lines, andthe evaluation of the crosses. There are many analytical methodsavailable to evaluate the result of a cross. The oldest and mosttraditional method of analysis is the observation of phenotypic traitsbut genotypic analysis may also be used.

Using PHACE in a Breeding Program

This invention is directed to methods for producing a maize plant bycrossing a first parent maize plant with a second parent maize plantwherein either the first or second parent maize plant is an inbred maizeplant of the line PHACE. The other parent may be any other maize plant,such as another inbred line or a plant that is part of a synthetic ornatural population. Any such methods using the inbred maize line PHACEare part of this invention: selfing, sibbing, backcrosses, massselection, pedigree breeding, bulk selection, hybrid production, crossesto populations, and the like. These methods are well known in the artand some of the more commonly used breeding methods are described below.Descriptions of breeding methods can also be found in one of severalreference books (e.g., Allard, Principles of Plant Breeding, 1960;Simmonds, Principles of Crop Improvement, 1979; Fehr, “Breeding Methodsfor Cultivar Development”, Production and Uses, 2^(nd) ed., Wilcoxeditor, 1987).

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such asPHACE and one other elite inbred line having one or more desirablecharacteristics that is lacking or which complements PHACE. If the twooriginal parents do not provide all the desired characteristics, othersources can be included in the breeding population. In the pedigreemethod, superior plants are selfed and selected in successive filialgenerations. In the succeeding filial generations the heterozygouscondition gives way to homogeneous lines as a result of self-pollinationand selection. Typically in the pedigree method of breeding, five ormore successive filial generations of selfing and selection ispracticed: F1→F2; F2→F3; F3→F4; F4→F₅, etc. After a sufficient amount ofinbreeding, successive filial generations will serve to increase seed ofthe developed inbred. Preferably, the inbred line comprises homozygousalleles at about 95% or more of its loci.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding to modify PHACEand a hybrid that is made using the modified PHACE. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one line, the donor parent, to aninbred called the recurrent parent, which has overall good agronomiccharacteristics yet lacks that desirable trait or traits. However, thesame procedure can be used to move the progeny toward the genotype ofthe recurrent parent but at the same time retain many components of thenon-recurrent parent by stopping the backcrossing at an early stage andproceeding with selfing and selection. For example, an F1, such as acommercial hybrid, is created. This commercial hybrid may be backcrossedto one of its parent lines to create a BC1 or BC2. Progeny are selfedand selected so that the newly developed inbred has many of theattributes of the recurrent parent and yet several of the desiredattributes of the non-recurrent parent. This approach leverages thevalue and strengths of the recurrent parent for use in new hybrids andbreeding.

Therefore, an embodiment of this invention is a method of making abackcross conversion of maize inbred line PHACE, comprising the steps ofcrossing a plant of maize inbred line PHACE with a donor plantcomprising a mutant gene or transgene conferring a desired trait,selecting an F1 progeny plant comprising the mutant gene or transgeneconferring the desired trait, and backcrossing the selected F1 progenyplant to a plant of maize inbred line PHACE. This method may furthercomprise the step of obtaining a molecular marker profile of maizeinbred line PHACE and using the molecular marker profile to select for aprogeny plant with the desired trait and the molecular marker profile ofPHACE. In the same manner, this method may be used to produce an F1hybrid seed by adding a final step of crossing the desired traitconversion of maize inbred line PHACE with a different maize plant tomake F1 hybrid maize seed comprising a mutant gene or transgeneconferring the desired trait.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. PHACE is suitable for use in a recurrentselection program. The method entails individual plants crosspollinating with each other to form progeny. The progeny are grown andthe superior progeny selected by any number of selection methods, whichinclude individual plant, half-sib progeny, full-sib progeny, selfedprogeny and topcrossing. The selected progeny are cross pollinated witheach other to form progeny for another population. This population isplanted and again superior plants are selected to cross pollinate witheach other. Recurrent selection is a cyclical process and therefore canbe repeated as many times as desired. The objective of recurrentselection is to improve the traits of a population. The improvedpopulation can then be used as a source of breeding material to obtaininbred lines to be used in hybrids or used as parents for a syntheticcultivar. A synthetic cultivar is the resultant progeny formed by theintercrossing of several selected inbreds.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection seeds fromindividuals are selected based on phenotype and/or genotype. Theseselected seeds are then bulked and used to grow the next generation.Bulk selection requires growing a population of plants in a bulk plot,allowing the plants to self-pollinate, harvesting the seed in bulk andthen using a sample of the seed harvested in bulk to plant the nextgeneration. Instead of self pollination, directed pollination could beused as part of the breeding program.

Mutation Breeding

Mutation breeding is one of many methods that could be used to introducenew traits into PHACE. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation; such as X-rays, Gamma rays (e.g.cobalt 60 or cesium 137), neutrons, (product of nuclear fission byuranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm), or chemical mutagens (suchas base analogues (5-bromo-uracil), related compounds (8-ethoxycaffeine), antibiotics (streptonigrin), alkylating agents (sulfurmustards, nitrogen mustards, epoxides, ethylenamines, sulfates,sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, oracridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques, such as backcrossing. Details of mutation breedingcan be found in “Principals of Cultivar Development” Fehr, 1993Macmillan Publishing Company the disclosure of which is incorporatedherein by reference. In addition, mutations created in other lines maybe used to produce a backcross conversion of PHACE that comprises suchmutation.

Breeding with Molecular Markers

Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats(SSRs) and Single Nucleotide Polymorphisms (SNPs), may be used in plantbreeding methods utilizing PHACE.

Isozyme Electrophoresis and RFLPs as discussed in Lee, M., “Inbred Linesof Maize and Their Molecular Markers,” The Maize Handbook,(Springer-Verlag, New York, Inc. 1994, at 423–432), have been widelyused to determine genetic composition. Isozyme Electrophoresis has arelatively low number of available markers and a low number of allelicvariants among maize inbreds. RFLPs allow more discrimination becausethey have a higher degree of allelic variation in maize and a largernumber of markers can be found. Both of these methods have been eclipsedby SSRs as discussed in Smith et al., “An evaluation of the utility ofSSR loci as molecular markers in maize (Zea mays L.): comparisons withdata from RFLPs and pedigree”, Theoretical and Applied Genetics (1997)vol. 95 at 163–173 and by Pejic et al., “Comparative analysis of geneticsimilarity among maize inbreds detected by RFLPs, RAPDs, SSRs, andAFLPs,” Theoretical and Applied Genetics (1998) at 1248–1255incorporated herein by reference. SSR technology is more efficient andpractical to use than RFLPs; more marker loci can be routinely used andmore alleles per marker locus can be found using SSRs in comparison toRFLPs. Single Nucleotide Polymorphisms may also be used to identify theunique genetic composition of the invention and progeny lines retainingthat unique genetic composition. Various molecular marker techniques maybe used in combination to enhance overall resolution.

Maize DNA molecular marker linkage maps have been rapidly constructedand widely implemented in genetic studies. One such study is describedin Boppenmaier, et al., “Comparisons among strains of inbreds forRFLPs”, Maize Genetics Cooperative Newsletter, 65:1991, pg. 90, isincorporated herein by reference.

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants. Itcan also be used to reduce the number of crosses back to the recurrentparent needed in a backcrossing program. The use of molecular markers inthe selection process is often called genetic marker enhanced selection.

Production of Double Haploids

The production of double haploids can also be used for the developmentof inbreds in the breeding program. For example, an F1 hybrid for whichPHACE is a parent can be used to produce double haploid plants. Doublehaploids are produced by the doubling of a set of chromosomes (1N) froma heterozygous plant to produce a completely homozygous individual. Forexample, see Wan et al., “Efficient Production of Doubled Haploid PlantsThrough Colchicine Treatment of Anther-Derived Maize Callus”,Theoretical and Applied Genetics, 77:889–892, 1989 and US2003/0005479.This can be advantageous because the process omits the generations ofselfing needed to obtain a homozygous plant from a heterozygous source.

Thus, an embodiment of this invention is a process for making asubstantially homozygous PHACE progeny plant by producing or obtaining aseed from the cross of PHACE and another maize plant and applying doublehaploid methods to the F1 seed or F1 plant or to any successive filialgeneration. Such methods decrease the number of generations required toproduce an inbred with similar genetics or characteristics to PHACE. SeeBernardo, R. and Kahler, A. L., Theor. Appl. Genet. 102:986–992, 2001.

In particular, a process of making seed retaining the molecular markerprofile of maize inbred line PHACE is contemplated, such processcomprising obtaining or producing F1 hybrid seed for which maize inbredline PHACE is a parent, inducing doubled haploids to create progenywithout the occurrence of meiotic segregation, obtaining the molecularmarker profile of maize inbred line PHACE, and selecting progeny thatretain the molecular marker profile of PHACE.

Use of PHACE in Tissue Culture

This invention is also directed to the use of PHACE in tissue culture.As used herein, the term “tissue culture” includes plant protoplasts,plant cell tissue culture, cultured microspores, plant calli, plantclumps, and the like. As used herein, phrases such as “growing the seed”or “grown from the seed” include embryo rescue, isolation of cells fromseed for use in tissue culture, as well as traditional growing methods.

Duncan, Williams, Zehr, and Widholm, Planta (1985) 165:322–332 reflectsthat 97% of the plants cultured that produced callus were capable ofplant regeneration. Subsequent experiments with both inbreds and hybridsproduced 91% regenerable callus that produced plants. In a further studyin 1988, Songstad, Duncan & Widholm in Plant Cell Reports (1988),7:262–265 reports several media additions that enhance regenerability ofcallus of two inbred lines. Other published reports also indicated that“nontraditional” tissues are capable of producing somatic embryogenesisand plant regeneration. K. P. Rao, et al., Maize Genetics CooperationNewsleffer, 60:64–65 (1986), refers to somatic embryogenesis from glumecallus cultures and B. V. Conger, et al., Plant Cell Reports, 6:345–347(1987) indicates somatic embryogenesis from the tissue cultures of maizeleaf segments. Thus, it is clear from the literature that the state ofthe art is such that these methods of obtaining plants are, and were,“conventional” in the sense that they are routinely used and have a veryhigh rate of success.

Tissue culture of maize, including tassel/anther culture, is describedin U.S. 2002/0062506A1 and European Patent Application, publication160,390, each of which are incorporated herein by reference for thispurpose. Maize tissue culture procedures are also described in Green andRhodes, “Plant Regeneration in Tissue Culture of Maize,” Maize forBiological Research (Plant Molecular Biology Association,Charlottesville, Va. 1982, at 367–372) and in Duncan, et al., “TheProduction of Callus Capable of Plant Regeneration from Immature Embryosof Numerous Zea Mays Genotypes,” 165 Planta 322–332 (1985). Thus,another aspect of this invention is to provide cells which upon growthand differentiation produce maize plants having the genotype and/orphysiological and morphological characteristics of inbred line PHACE.

Progeny Plants

All plants produced by the use of the methods described herein and thatretain the unique genetic or trait combinations of PHACE are within thescope of the invention. Progeny of the breeding methods described hereinmay be characterized in any number of ways, such as by traits retainedin the progeny, pedigree and/or molecular markers. Combinations of thesemethods of characterization may be used.

Breeder's of ordinary skill in the art have developed the concept of an“essentially derived variety”, which is defined in 7 U.S.C. § 2104(a)(3)of the Plant Variety Protection Act and is hereby incorporated byreference. Varieties and plants that are essentially derived from PHACEare within the scope of the invention.

Pedigree is a method used by breeders of ordinary skill in the art todescribe the varieties. Varieties that are more closely related bypedigree are likely to share common genotypes and combinations ofphenotypic characteristics. All breeders of ordinary skill in the artmaintain pedigree records of their breeding programs. These pedigreerecords contain a detailed description of the breeding process,including a listing of all parental lines used in the breeding processand information on how such line was used. One embodiment of thisinvention is progeny plants and parts thereof with at least one ancestorthat is PHACE, and more specifically, where the pedigree of the progenyincludes 1, 2, 3, 4, and/or 5 or less breeding crosses to a maize plantother than PHACE or a plant that has PHACE as a parent or otherprogenitor. A breeder of ordinary skill in the art would know if PHACEwere used in the development of a progeny line, and would also know howmany crosses to a line other than PHACE or line with PHACE as a parentor other progenitor were made in the development of any progeny line.

Molecular markers also provide a means by which those of ordinary skillin the art characterize the similarity or differences of two lines.Using the breeding methods described herein, one can develop individualplants, plant cells, and populations of plants that retain at least 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 99.5% genetic contribution from inbred line PHACE,as measured by either percent identity or percent similarity. Inpedigree analysis the percentage genetic contribution may not beactually known, but on average 50% of the starting germplasm would beexpected to be passed to the progeny line after one cross to anotherline, 25% after another cross to a different line, and so on. Withbackcrossing, the expected contribution of PHACE after 2, 3, 4 and 5doses (or 1, 2, 3 and 4 backcrosses) would be 75%, 87.5%, 93.75% and96.875% respectively. Actual genetic contribution may be much higherthan the genetic contribution expected by pedigree, especially ifmolecular markers are used in selection. Molecular markers could also beused to confirm and/or determine the pedigree of the progeny line.

Traits are also used by those of ordinary skill in the art tocharacterize progeny. Traits are commonly evaluated at a significancelevel, such as a 1%, 5% or 10% significance level, when measured inplants grown in the same environmental conditions. For example, abackcross conversion of PHACE may be characterized as having the samemorphological and physiological traits as PHACE. The traits used forcomparison may be any or all of the traits shown in Table 1, Table 3,Table 4 or Table 5.

A breeder will commonly work to combine a specific trait of anundeveloped variety of the species, such as a high level of resistanceto a particular disease, with one or more of the elite agronomiccharacteristics (yield, maturity, plant size, lodging resistance, etc.)needed for use as a commercial variety. This combination, oncedeveloped, provides a valuable source of new germplasm for furtherbreeding. For example, it may take 10–15 years and significant effort toproduce such a combination, yet progeny may be developed that retainthis combination in as little as 2–5 years and with much less effort.

Specific Embodiments

Specific methods and products produced using inbred line PHACE in plantbreeding are discussed in the following sections. The methods outlinedare described in detail by way of illustration and example for purposesof clarity and understanding. However, it will be obvious that certainchanges and modifications may be practiced within the scope of theinvention.

One method for producing a line derived from inbred line PHACE is asfollows. One of ordinary skill in the art would produce or obtain a seedfrom the cross between inbred line PHACE and another variety of maize,such as an elite inbred variety. The F1 seed derived from this crosswould be grown to form a homogeneous population. The F1 seed wouldcontain essentially all of the alleles from variety PHACE andessentially all of the alleles from the other maize variety. The F1nuclear genome would be made-up of 50% variety PHACE and 50% of theother elite variety. The F1 seed would be grown and allowed to self,thereby forming F2 seed. On average the F2 seed would have derived 50%of its alleles from variety PHACE and 50% from the other maize variety,but many individual plants from the population would have a greaterpercentage of their alleles derived from PHACE (Wang J. and R. Bernardo,2000, Crop Sci. 40:659–665 and Bernardo, R. and A. L. Kahler, 2001,Theor. Appl. Genet 102:986–992). The molecular markers of PHACE could beused to select and retain those lines with high similarity to PHACE. TheF2 seed would be grown and selection of plants would be made based onvisual observation, markers and/or measurement of traits. The traitsused for selection may be any PHACE trait described in thisspecification, including the inbred per se maize PHACE traits describedherein under the detailed description of inbred PHACE. Such traits mayalso be the good general or specific combining ability of PHACE,including its ability to produce hybrids with the approximate maturityand/or hybrid combination traits described herein under the detaileddescription of inbred PHACE. The PHACE progeny plants that exhibit oneor more of the desired PHACE traits, such as those listed herein, wouldbe selected and each plant would be harvested separately. This F3 seedfrom each plant would be grown in individual rows and allowed to self.Then selected rows or plants from the rows would be harvestedindividually. The selections would again be based on visual observation,markers and/or measurements for desirable traits of the plants, such asone or more of the desirable PHACE traits listed herein. The process ofgrowing and selection would be repeated any number of times until aPHACE progeny inbred plant is obtained. The PHACE progeny inbred plantwould contain desirable traits derived from inbred plant PHACE, some ofwhich may not have been expressed by the other maize variety to whichinbred line PHACE was crossed and some of which may have been expressedby both maize varieties but now would be at a level equal to or greaterthan the level expressed in inbred variety PHACE. However, in each casethe resulting progeny line would benefit from the efforts of theinventor(s), and would not have existed but for the inventor(s) work increating PHACE. The PHACE progeny inbred plants would have, on average,50% of their nuclear genes derived from inbred line PHACE, but manyindividual plants from the population would have a greater percentage oftheir alleles derived from PHACE. This breeding cycle, of crossing andselfing, and optional selection, may be repeated to produce anotherpopulation of PHACE progeny maize plants with, on average, 25% of theirnuclear genes derived from inbred line PHACE, but, again, manyindividual plants from the population would have a greater percentage oftheir alleles derived from PHACE. This process can be repeated for athird, fourth, fifth, sixth, seventh or more breeding cycles. Anotherembodiment of the invention is a PHACE progeny plant that has receivedthe desirable PHACE traits listed herein through the use of PHACE, whichtraits were not exhibited by other plants used in the breeding process.

Therefore, an embodiment of this invention is a PHACE progeny maizeplant, wherein at least one ancestor of said PHACE progeny maize plantis the maize plant or plant part of PHACE, and wherein the pedigree ofsaid PHACE progeny maize plant is within two breeding crosses of PHACEor a plant that has PHACE as a parent. The progeny plants, parts andplant cells produced from PHACE may be further characterized as having apercent marker similarity or identity with PHACE as described herein.

The previous example can be modified in numerous ways, for instanceselection may or may not occur at every selfing generation, selectionmay occur before or after the actual self-pollination process occurs, orindividual selections may be made by harvesting individual ears, plants,rows or plots at any point during the breeding process described. Doublehaploid breeding methods may be used at any step in the process. Insteadof selfing out of the hybrid produced from the inbred, one could firstcross the hybrid to either a parent line or a different inbred, and thenself out of that cross.

The population of plants produced at each and any cycle of breeding isalso an embodiment of the invention, and on average each such populationwould predictably consist of plants containing approximately 50% of itsgenes from inbred line PHACE in the first breeding cycle, 25% of itsgenes from inbred line PHACE in the second breeding cycle, 12.5% of itsgenes from inbred line PHACE in the third breeding cycle, 6.25% in thefourth breeding cycle, 3.125% in the fifth breeding cycle, and so on.However, in each case the use of PHACE provides a substantial benefit.The linkage groups of PHACE would be retained in the progeny lines, andsince current estimates of the maize genome size is about 50,000–80,000genes (Xiaowu, Gai et al., Nucleic Acids Research, 2000, Vol. 28, No. 1,94–96), in addition to non-coding DNA that impacts gene expression, itprovides a significant advantage to use PHACE as starting material toproduce a line that retains desired genetics or traits of PHACE.

Therefore, an embodiment of the invention is a process for making apopulation of PHACE progeny inbred maize plants comprising obtaining orproducing a first generation progeny maize seed comprising the plant ofPHACE as a parent, growing said first generation progeny maize seed toproduce first generation maize plants and obtaining self or sibpollinated seed from said first generation maize plants, and growing theself or sib pollinated seed to obtain a population of PHACE progenyinbred maize plants.

The population of PHACE progeny inbred maize plants produced by thismethod are also embodiments of the invention, and such population as awhole will retain the expected genetic contribution of PHACE. An inbredline selected from the population of PHACE progeny inbred maize plantsproduced by this method is an embodiment, and such line may be furthercharacterized by its molecular marker identity or similarity to PHACE.

In this manner, the invention also encompasses a process for making aPHACE inbred progeny maize plant comprising the steps of obtaining orproducing a first generation progeny maize seed wherein a parent of saidfirst generation progeny maize seed is a PHACE plant, growing said firstgeneration progeny maize seed to produce a first generation maize plantand obtaining self or sib pollinated seed from said first generationmaize plant, and producing successive filial generations to obtain aPHACE inbred progeny maize plant. Also an embodiment of this inventionis the first breeding cycle inbred PHACE maize plant produced by thismethod.

Crosses to Other Species

The utility of inbred maize line PHACE also extends to crosses withother species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Coix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Potentially suitable for crosses with PHACE may be the variousvarieties of grain sorghum, Sorghum bicolor (L.) Moench.

INDUSTRIAL APPLICABILITY

Maize is used as human food, livestock feed, and as raw material inindustry. The food uses of maize, in addition to human consumption ofmaize kernels, include both products of dry- and wet-milling industries.The principal products of maize dry milling are grits, meal and flour.The maize wet-milling industry can provide maize starch, maize syrups,and dextrose for food use. Maize oil is recovered from maize germ, whichis a by-product of both dry- and wet-milling industries.

Maize, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry.

Industrial uses of maize include production of ethanol, maize starch inthe wet-milling industry and maize flour in the dry-milling industry.The industrial applications of maize starch and flour are based onfunctional properties, such as viscosity, film formation, adhesiveproperties, and ability to suspend particles. The maize starch and flourhave application in the paper and textile industries. Other industrialuses include applications in adhesives, building materials, foundrybinders, laundry starches, explosives, oil-well muds, and other miningapplications.

Plant parts other than the grain of maize are also used in industry: forexample, stalks and husks are made into paper and wallboard and cobs areused for fuel and to make charcoal.

The seed of inbred maize line PHACE, the plant produced from the inbredseed, the hybrid maize plant produced from the crossing of the inbred,hybrid seed, and various parts of the hybrid maize plant and transgenicversions of the foregoing, can be utilized for human food, livestockfeed, and as a raw material in industry.

Deposits

Applicants have made a deposit of at least 2500 seeds of Inbred MaizeLine PHACE with the American Type Culture Collection (ATCC), Manassas,Va. 20110 USA, ATCC Deposit No PTA-6341. The seeds deposited with theATCC on Nov. 30, 2004 were taken from the deposit maintained by PioneerHi-Bred International, Inc., 7100 NW 62^(nd) Avenue, Johnston, Iowa50131-1000 since prior to the filing date of this application. Access tothis deposit will be available during the pendency of the application tothe Commissioner of Patents and Trademarks and persons determined by theCommissioner to be entitled thereto upon request. Upon allowance of anyclaims in the application, the Applicant(s) will make the depositavailable to the public pursuant to 37 CFR 1.808. This deposit of theInbred Maize Line PHACE will be maintained in the ATCC depository, whichis a public depository, for a period of 30 years, or 5 years after themost recent request, or for the enforceable life of the patent,whichever is longer, and will be replaced if it becomes nonviable duringthat period. Additionally, Applicant has or will satisfy all therequirements of 37 C.F.R. §§1.801–1.809, including providing anindication of the viability of the sample upon deposit. Applicant has noauthority to waive any restrictions imposed by law on the transfer ofbiological material or its transportation in commerce. Applicant doesnot waive any infringement of their rights granted under this patent orunder the Plant Variety Protection Act (7 USC 2321 et seq.). U.S. PlantVariety Protection of Inbred Maize Line PHACE has been applied for.Unauthorized seed multiplication is prohibited.

Tables

TABLE 1 VARIETY DESCRIPTION INFORMATION VARIETY = PHACE AVG STDEV N 1.TYPE: (Describe intermediate types in comments section) 1 = Sweet, 2 =Dent, 3 = Flint, 4 = Flour, 5 = Pop and 2 6 = Ornamental. Comments: DentLike 2. MATURITY: DAYS HEAT UNITS Days H. Units Emergence to 50% ofplants in silk 62 1,338 Emergence to 50% of plants in pollen shed 621,327 10% to 90% pollen shed 2 53 50% Silk to harvest at 25% moisture 3.PLANT: Plant Height (to tassel tip) (cm) 237.7 14.39 15 Ear Height (tobase of top ear node) (cm) 97.4 13.54 15 Length of Top Ear Internode(cm) 16.2 2.51 15 Average Number of Tillers per Plant 0.3 0.43 3 AverageNumber of Ears per Stalk 1.2 0.20 3 Anthocyanin of Brace Roots: 1 =Absent, 2 = Faint, 2 3 = Moderate, 4 = Dark 4. LEAF: Width of Ear NodeLeaf (cm) 8.7 0.46 15 Length of Ear Node Leaf (cm) 79.9 2.92 15 Numberof Leaves above Top Ear 6.5 0.64 15 Leaf Angle: (at anthesis, 2nd leafabove ear to 26.7 3.10 15 stalk above leaf) (Degrees) *Leaf Color: V.Dark Green Munsell: 7.5GY36 Leaf Sheath Pubescence: 1 = none to 9 = likepeach fuzz 3 5. TASSEL: Number of Primary Lateral Branches 6.9 1.79 15Branch Angle from Central Spike 27.6 11.76 15 Tassel Length: (frompeduncle node to tassel tip), (cm). 55.2 3.76 15 Pollen Shed: 0 = malesterile, 9 = heavy shed 5 *Anther Color: Pale Yellow Munsell: 2.5Y8.56*Glume Color: Med. Green Munsell: 5GY66 *Bar Glumes (glume bands): 1 =absent, 2 = present 1 Peduncle Length: (from top leaf node to lowerflorets or 21.8 2.93 15 branches), (cm). 6a. EAR (Unhusked ear) *Silkcolor: Light Red Munsell: 2.5R58 (3 days after silk emergence) *Freshhusk color: Med. Green Munsell: 5GY78 *Dry husk color: White Munsell:10YR92 (65 days after 50% silking) Ear position at dry husk stage: 1 =upright, 2 = horizontal, 3 3 = pendant Husk Tightness: (1 = very loose,9 = very tight) 7 Husk Extension (at harvest): 1 = short(ears exposed),2 2 = medium (<8 cm), 3 = long (8–10 cm), 4 = v. long (>10 cm) 6b. EAR(Husked ear data) Ear Length (cm): 15.6 0.63 15 Ear Diameter atmid-point (mm) 38.2 1.08 15 Ear Weight (gm): 99.4 12.61 15 Number ofKernel Rows: 13.9 1.41 15 Kernel Rows: 1 = indistinct, 2 = distinct 2Row Alignment: 1 = straight, 2 = slightly curved, 3 = spiral 2 ShankLength (cm): 8.3 1.63 15 Ear Taper: 1 = slight cylind., 2 = average, 3 =extreme 2 7. KERNEL (Dried): Kernel Length (mm): 10.1 0.46 15 KernelWidth (mm): 7.4 0.63 15 Kernel Thickness (mm): 3.9 0.52 15 Round Kernels(shape grade) (%) 12.6 2.77 3 Aleurone Color Pattern: 1 = homozygous, 2= segregating 1 *Aleurone Color: Yellow Munsell: 10YR814 *Hard Endo.Color: Yellow Munsell: 10YR612 Endosperm Type: 3 1 = sweet (su1), 2 =extra sweet (sh2), 3 = normal starch, 4 = high amylose starch, 5 = waxystarch, 6 = high protein, 7 = high lysine, 8 = super sweet (se), 9 =high oil, 10 = other Weight per 100 Kernels (unsized sample) (gm): 18.72.52 3 8. COB: *Cob Diameter at mid-point (mm): 20.8 0.86 15 *Cob Color:Pink Orange Munsell: 2.5YR48 10. DISEASE RESISTANCE: (Rate from 1 =most-susceptable to 9 = most-resistant. Leave blank if not tested, leaverace or strain options blank if polygenic.) A. LEAF BLIGHTS, WILTS, ANDLOCAL INFECTION DISEASES Anthracnose Leaf Blight (Colletotrichumgraminicola) Common Rust (Puccinia sorghi) Common Smut (Ustilago maydis)6 Eyespot (Kabatiella zeae) Gross's Wilt (Clavibacter michiganense spp.nebraskense) 5 Gray Leaf Spot (Cercospora zeae-maydis) HelminthosporiumLeaf Spot (Bipolaris zeicola) Race: 4 Northern Leaf Blight (Exserohilumturcicum) Race: 6 Southern Leaf Blight (Bipolaris maydis) Race: SouthernRust (Puccinia polysora) Stewart's Wilt (Erwinia stewartii) Other(Specify):            B. SYSTEMIC DISEASES Corn Lethal Necrosis (MCMVand MDMV) 7 Head Smut (Sphacelotheca reiliana) Maize Chlorotic DwarfVirus (MDV) Maize Chlorotic Mottle Virus (MCMV) 5 Maize Dwarf MosaicVirus (MDMV) Sorghum Downy Mildew of Corn (Peronosclerospora sorghi)Other (Specify):            C. STALK ROTS 6 Anthracnose Stalk Rot(Colletotrichum graminicola) Diplodia Stalk Rot (Stenocarpella maydis)Fusarium Stalk Rot (Fusarium moniliforme) Gibberella Stalk Rot(Gibberella zeae) Other (Specify):            D. EAR AND KERNEL ROTSAspergillus Ear and Kernel Rot (Aspergillus flavus) Diplodia Ear Rot(Stenocarpella maydis) Fusarium Ear and Kernel Rot (Fusariummoniliforme) Gibberella Ear Rot (Gibberella zeae) Other (Specify):           11. INSECT RESISTANCE: (Rate from 1 = most-suscept. to 9 =most-resist., leave blank if not tested.) Corn Worm (Helicoverpa zea)    Leaf Feeding     Silk Feeding     Ear Damage Corn Leaf Aphid(Rophalosiphum maydis) Corn Sap Beetle (Capophilus dimidiatus) EuropeanCorn Borer (Ostrinia nubilalis) 1st. Generation (Typically whorl leaffeeding) 2nd. Generation (Typically leaf sheath-collar feeding)    Stalk Tunneling     cm tunneled/plant Fall armyworm (Spodopterafruqiperda)     Leaf Feeding     Silk Feeding     mg larval wt. MaizeWeevil (Sitophilus zeamaize) Northern Rootworm (Diabrotica barberi)Southern Rootworm (Diabrotica undecimpunctata) Southwestern Corn Borer(Diatreaea grandiosella)     Leaf Feeding     Stalk Tunneling     cmtunneled/plant Two-spotted Spider Mite (Tetranychus utricae) WesternRootworm (Diabrotica virgifrea virgifrea) Other (Specify):           12. AGRONOMIC TRAITS: Staygreen (at 65 days after anthesis; rate from1-worst to 9-excellent) % Dropped Ears (at 65 days after anthesis) %Pre-anthesis Brittle Snapping % Pre-anthesis Root Lodging %Post-anthesis Root Lodging (at 65 days after anthesis) % Post-anthesisStalk Lodging 7,196.0 Kg/ha (Yield at 12–13% grain moisture) *MunsellGlossy Book of Color, (A standard color reference). Kollmorgen Inst.Corp. New Windsor, NY.

TABLE 2 SSR PROFILE OF PHACE Bin # Marker Name Base Pairs 1.01 UMC1170229.99 1.01 UMC2225 300.48 1.02 BNLG1007 132.69 1.03 BNLG1203 306.4 1.04BNLG1016 255.77 1.04 BNLG2238 204.46 1.06 UMC1035 233.32 1.08 BNLG2228271.66 1.09 PHI011 226.64 1.09 UMC1306 147.65 1.11 PHI227562 319.53 2UMC1419 110.05 2.01 UMC1227 139.44 2.01 UMC1378 121.68 2.02 UMC1265293.86 2.05 BNLG1909 295.74 2.05 PHI014 432.23 2.05 UMC1202 248.96 2.05UMC1506 168.48 2.06 BNLG1036 194.95 2.06 UMC1299 143.36 2.07 BNLG1927198.54 2.07 PHI251315 124.05 2.07 UMC1560 136.56 2.08 BNLG1258 217.72.08 BNLG1940 247.8 2.08 UMC1049 128.56 2.08 UMC1889 113.38 3.04BNLG1113 134.5 3.04 BNLG1638 142.21 3.04 UMC1036 325.35 3.06 PHI07080.98 3.1 UMC1136 146.33 4.05 UMC1142 155.17 4.05 UMC1303 127.11 4.05UMC1317 113.76 4.05 UMC1346 93.63 4.05 UMC1382 159.52 4.05 UMC1390133.36 4.05 UMC1451 114.07 4.05 UMC1511 166.16 4.05 UMC1548 159.53 4.05UMC1702 101.02 4.05 UMC1791 157.03 4.05 UMC1851 114.22 4.05 UMC189687.87 4.05 UMC1964 146.37 4.05 UMC2054 151.1 4.05 UMC2055 87.43 4.05UMC2061 125.23 4.06 BNLG1621 171.1 4.06 BNLG2291 160.65 4.06 MMC0371266.83 4.06 UMC1662 111.92 4.06 UMC1869 154.49 4.06 UMC1945 113.43 4.06UMC2027 116.54 4.07 BNLG1784 231.4 4.07 UMC1620 144.16 4.08 DUPSSR28128.38 4.08 PHI093 280.75 4.08 UMC1086 105.92 4.08 UMC1371 163.21 4.08UMC1667 154.52 4.08 UMC1808 139.6 4.08 UMC1834 161.33 4.08 UMC1856189.51 4.08 UMC1871 128.62 4.08 UMC1899 111.71 4.08 UMC2041 171.25 4.08UMC2187 87.02 4.08 UMC2188 168.06 4.09 BNLG1565 204.82 4.09 UMC1101137.41 4.09 UMC1328 161.28 4.09 UMC1574 167.23 4.09 UMC1631 139.72 4.09UMC1650 139.58 4.09 UMC1820 138.94 4.09 UMC1940 134.49 4.09 UMC1999131.31 4.09 UMC2046 143.83 4.09 UMC2139 133.81 5.01 PHI024 367.8 5.03UMC1352 148.67 5.04 UMC1424 294.45 5.07 BNLG1346 189.4 6 UMC1002 123.646 UMC1143 233.38 6.01 BNLG1422 216.1 6.01 PHI077 124.79 6.01 UMC1133219.99 6.01 UMC1195 134.48 6.01 UMC1596 103.14 6.01 UMC2056 163.88 6.02UMC1083 345.34 6.02 UMC1628 160.36 6.04 UMC1857 146.13 6.04 UMC1918163.25 6.04 UMC2006 174.02 6.04 UMC2317 123.73 6.05 UMC1020 136.57 6.05UMC1045 184.78 6.05 UMC1250 265.24 6.05 UMC1314 346.57 6.05 UMC1341351.36 6.06 UMC1474 220.9 6.06 UMC1762 307.26 6.06 UMC1859 106.71 6.06UMC1912 169.27 6.07 UMC1127 231.44 6.07 UMC1248 122.16 6.07 UMC1296141.7 6.07 UMC1621 211.45 6.07 UMC1653 252.16 7 BNLG2132 200.36 7UMC1241 120.99 7 UMC1642 140.75 7.01 BNLG1292 121.51 7.01 PHI057 415.597.01 UMC1270 255.16 7.01 UMC1632 150.12 7.02 BNLG1094 145.02 7.02DUPSSR9 116.56 7.02 UMC1393 323.22 7.02 UMC1401 163.19 7.02 UMC1433135.22 7.02 UMC1978 115.23 7.03 BNLG1070 145.49 7.03 BNLG155 251.11 7.03UMC1015 93.12 7.03 UMC1301 335.08 7.03 UMC1450 126.71 7.03 UMC1456115.52 7.03 UMC1660 204.58 7.03 UMC1713 139.57 7.03 UMC1865 145.07 7.03UMC1888 145.8 7.04 UMC1125 190.27 7.04 UMC1295 247.86 7.04 UMC1412151.81 7.04 UMC1710 235.29 7.04 UMC1768 148.99 7.04 UMC1782 321.7 7.04UMC1944 147.65 7.05 UMC1154 280.68 7.05 UMC1407 359.59 7.06 UMC174098.29 8.01 UMC1139 305.96 8.01 UMC1483 310.42 8.01 UMC1786 332.21 8.01UMC2042 107.83 8.02 UMC1034 130.69 8.02 UMC1304 251.47 8.02 UMC1428321.36 8.02 UMC1790 157.73 8.02 UMC1872 152.85 8.02 UMC1913 161.98 8.02UMC1974 486.32 8.03 BNLG1863 244.51 8.03 UMC1457 341.09 8.03 UMC1470337.11 8.03 UMC1735 95.2 8.03 UMC1741 162.77 8.03 UMC1807 104.25 8.03UMC1904 155.28 8.03 UMC1910 170.74 8.03 UMC2057 151.48 8.04 UMC1172338.21 8.04 UMC1343 323.03 8.04 UMC1858 127.94 8.05 UMC1130 236.39 8.05UMC1141 306.87 8.05 UMC1263 348.26 8.05 UMC1562 209.97 8.05 UMC1864170.58 8.05 UMC1882 114.44 8.05 UMC1959 330.17 8.06 BNLG1031 288.07 8.06UMC1161 258.15 8.06 UMC1670 124.34 8.07 UMC1607 214.51 8.08 UMC1933113.55 9 UMC2393 212.5 9.01 BNLG1810 205.17 9.01 UMC1370 323.96 9.01UMC1588 316.99 9.01 UMC1958 231.69 9.02 UMC1131 362.48 9.02 UMC2219254.21 9.03 BNLG1217 210.32 9.03 GL15 161.37 9.03 PHI022 238.22 9.03UMC1191 212.97 9.03 UMC1267 338.22 9.03 UMC1377 216.07 9.03 UMC1614332.9 9.03 UMC1743 136.77 9.03 UMC2087 266.37 9.03 UMC2394 367.31 9.04MMP96 220.9 9.04 UMC1522 252.89 9.04 UMC1771 330.92 9.04 UMC2121 164.599.05 MMP179 161.31 9.05 UMC1078 353.45 9.05 UMC1357 257.23 9.05 UMC1417209.61 9.05 UMC1794 115.28 9.05 UMC2341 122.07 9.06 PHI448880 182.829.07 UMC1714 158.76 9.07 UMC2089 134.49 9.07 UMC2131 264.21 9.08 UMC1277236.86 9.08 UMC1505 142.19 10 UMC1293 160.12 10.01 UMC1318 218.35 10.01UMC2018 154.53 10.02 UMC1152 169.49 10.02 UMC1337 306.29 10.02 UMC1432126.01 10.02 UMC1576 153.29 10.02 UMC2034 132.95 10.02 UMC2069 351.8710.03 UMC1037 215.65 10.03 UMC1312 282.17 10.03 UMC1345 166.44 10.03UMC1367 327.29 10.03 UMC1381 210.26 10.03 UMC1666 151.42 10.03 UMC1739317.29 10.03 UMC1785 218 10.03 UMC1863 161.77 10.03 UMC1866 163.9 10.03UMC1962 126.23 10.03 UMC2016 129.33 10.03 UMC2067 165.34 10.04 UMC1054348.39 10.04 UMC1246 230.39 10.04 UMC1272 201.76 10.04 UMC1280 302.3410.04 UMC1330 340.6 10.04 UMC1487 338.86 10.04 UMC1507 315.89 10.04UMC1648 141.5 10.04 UMC1930 125.02 10.04 UMC2003 89.07 10.06 UMC1249233.65 10.07 BNLG1839 186.98 10.07 UMC1038 261.11 10.07 UMC1084 254.1910.07 UMC1176 339.04 10.07 UMC1640 107.77 10.07 UMC1645 166.03 10.07UMC2021 135.3

TABLE 3A PAIRED INBRED COMPARISON REPORT Variety #1: PHACE Variety #2:PH6ME EGRWTH ESTCNT TILLER GDUSHD GDUSLK TASSZ SCORE COUNT PCT GDU GDUSCORE Stat ABS ABS ABS ABS ABS ABS Mean1 6.2 26.1 21.8 141.1 143.5 5.0Mean2 5.8 25.9 4.7 146.9 149.5 4.8 Locs 18 18 12 62 62 54 Reps 18 18 1262 62 54 Diff 0.4 0.2 −17.0 −5.7 −6.0 0.3 Prob 0.104 0.766 0.002 0.0000.000 0.164 PLTHT EARHT STAGRN SCTGRN TEXEAR EARMLD CM CM SCORE SCORESCORE SCORE Stat ABS ABS ABS ABS ABS ABS Mean1 228.0 84.9 4.2 7.7 6.76.3 Mean2 216.1 86.7 3.5 6.9 7.0 7.3 Locs 50 7 20 9 3 4 Reps 50 7 20 9 34 Diff 11.9 −1.8 0.7 0.8 −0.3 −1.0 Prob 0.000 0.834 0.090 0.154 0.4230.092 BARPLT GLFSPT NLFBLT SLFBLT ANTROT FUSERS % NOT SCORE SCORE SCORESCORE SCORE Stat ABS ABS ABS ABS ABS ABS Mean1 97.4 4.7 4.0 6.3 3.5 5.5Mean2 93.1 3.7 5.5 4.3 2.5 6.4 Locs 28 11 1 2 1 6 Reps 28 14 2 3 2 8Diff 4.2 1.0 −1.5 2.0 1.0 −0.9 Prob 0.111 0.046 — 0.295 — 0.120 GIBERSDIPERS COMRST HD SMT ERTLDG ERTLPN SCORE SCORE SCORE % NOT % NOT % NOTStat ABS ABS ABS ABS ABS ABS Mean1 9.0 4.3 6.5 71.4 52.2 100.0 Mean2 7.55.5 6.5 57.7 66.7 72.5 Locs 1 2 2 1 1 4 Reps 2 3 2 2 1 4 Diff 1.5 −1.30.0 13.7 −14.5 27.5 Prob — 0.344 1.000 — — 0.288

TABLE 3B PAIRED INBRED COMPARISON REPORT Variety #1: PHACE Variety #2:PH24E YIELD YIELD TSTWT EGRWTH ESTCNT BU/A 56# BU/A 56# MST PCT LB/BUSCORE COUNT Stat ABS % MN ABS ABS ABS ABS Mean1 118.1 125.2 18.7 58.26.2 27.6 Mean2 101.1 106.4 18.1 57.0 5.8 25.3 Locs 10 10 11 6 12 18 Reps10 10 11 6 12 18 Diff 17.0 18.8 −0.6 1.2 0.4 2.3 Prob 0.002 0.002 0.5650.075 0.210 0.001 TILLER GDUSHD GDUSLK POLWT POLWT TASSZ PCT GDU GDUVALUE VALUE SCORE Stat ABS ABS ABS ABS % MN ABS Mean1 25.7 140.1 142.1164.7 98.1 4.9 Mean2 1.0 138.1 141.9 162.8 99.4 5.2 Locs 10 40 40 6 6 34Reps 10 40 40 12 12 34 Diff −24.7 2.0 0.2 1.9 −1.3 −0.3 Prob 0.003 0.0040.810 0.941 0.936 0.257 PLTHT EARHT STAGRN SCTGRN TEXEAR EARMLD CM CMSCORE SCORE SCORE SCORE Stat ABS ABS ABS ABS ABS ABS Mean1 227.0 71.14.9 8.1 7.0 7.0 Mean2 207.8 83.8 4.9 6.5 6.5 6.3 Locs 29 3 11 8 2 3 Reps29 3 11 8 2 3 Diff 19.3 −12.7 0.0 1.6 0.5 0.7 Prob 0.000 0.342 1.0000.010 0.500 0.529 BARPLT GLFSPT NLFBLT SLFBLT ANTROT FUSERS % NOT SCORESCORE SCORE SCORE SCORE Stat ABS ABS ABS ABS ABS ABS Mean1 97.6 5.3 5.05.5 3.5 6.4 Mean2 99.1 4.6 5.7 5.5 4.0 4.6 Locs 21 7 3 1 1 7 Reps 21 116 2 2 10 Diff −1.5 0.7 −0.7 0.0 −0.5 1.9 Prob 0.289 0.229 0.184 — —0.014 DIPERS COMRST ECB2SC CLDTST CLDTST KSZDCD SCORE SCORE SCORE PCTPCT PCT Stat ABS ABS ABS ABS % MN ABS Mean1 6.0 6.5 5.0 92.0 101.0 5.3Mean2 6.0 7.0 9.0 89.0 97.7 2.5 Locs 1 2 1 4 4 4 Reps 2 2 1 4 4 4 Diff0.0 −0.5 −4.0 3.0 3.3 2.8 Prob — 0.500 — 0.406 0.407 0.010 ERTLDG ERTLPNLRTLPN % NOT % NOT % NOT Stat ABS ABS ABS Mean1 52.2 100.0 100.0 Mean2100.0 99.7 85.0 Locs 1 3 1 Reps 1 3 2 Diff −47.8 0.3 15.0 Prob — 0.423 —

TABLE 4 GENERAL COMBINING ABILITY REPORT PHACE PRM Day ABS Mean 113 PRMDay ABS Reps 5520 PRMSHD Day ABS Mean 112 PRMSHD Day ABS Reps 3534 YIELDbu/a 56# ABS Mean 190.6 YIELD bu/a 56# ABS Reps 2884 YIELD bu/a 56# ABSYears 3 YIELD bu/a 56# % MN Mean 103 YIELD bu/a 56# % MN Reps 2884 MSTpct ABS Mean 21.7 MST pct ABS Reps 2906 MST pct % MN Mean 101.2 MST pct% MN Reps 2906 STLPCN % NOT % MN Mean 99 STLPCN % NOT % MN Reps 650STLLPN % NOT % MN Mean 98 STLLPN % NOT % MN Reps 1108 ERTLPN % NOT % MNMean 106 ERTLPN % NOT % MN Reps 235 LRTLPN % NOT % MN Mean 102 LRTLPN %NOT % MN Reps 391 TSTWT lb/bu % MN Mean 99.6 TSTWT lb/bu % MN Reps 2115STKCNT count % MN Mean 100 STKCNT count % MN Reps 4752 PLTHT in % MNMean 101 PLTHT in % MN Reps 1057 EARHT in % MN Mean 102 EARHT in % MNReps 1014 BRTSTK % NOT % MN Mean 100 BRTSTK % NOT % MN Reps 283 GLFSPTscore ABS Mean 5 GLFSPT score ABS Reps 270 STAGRN score ABS Mean 5STAGRN score ABS Reps 582 HSKCVR score ABS Mean 6 HSKCVR score ABS Reps146

TABLE 5A INBREDS IN HYBRID COMBINATION REPORT Variety #1: HYBRIDCONTAINING PHACE Variety #2: 33M54 YIELD YIELD EGRWTH ESTCNT GDUSHD BU/A56# BU/A 56# MST PCT SCORE COUNT GDU Stat ABS % MN % MN % MN % MN % MNMean1 188.9 105.8 106.3 101.9 103.3 99.2 Mean2 179.2 100.1 103.8 93.396.0 100.8 Locs 109 109 110 16 11 23 Reps 119 119 120 18 15 30 Diff 9.75.7 −2.5 8.7 7.3 −1.5 Prob 0.000 0.000 0.001 0.250 0.003 0.000 GDUSLKSTKCNT PLTHT EARHT STAGRN STKLDG GDU COUNT CM CM SCORE % NOT Stat % MN %MN % MN % MN % MN % MN Mean1 99.8 100.3 100.5 102.6 118.4 100.8 Mean2100.8 99.3 97.3 94.2 106.7 104.0 Locs 19 193 32 30 29 1 Reps 25 281 3832 32 1 Diff −1.0 1.0 3.2 8.4 11.7 −3.2 Prob 0.031 0.029 0.006 0.0000.046 — ABTSTK TSTWT GLFSPT NLFBLT SLFBLT GOSWLT % NOT LB/BU SCORE SCORESCORE SCORE Stat % MN ABS ABS ABS ABS ABS Mean1 109.6 56.0 4.8 4.1 5.27.0 Mean2 102.7 57.4 5.9 4.5 7.0 8.0 Locs 3 75 9 7 5 1 Reps 15 81 13 138 2 Diff 6.9 −1.4 −1.1 −0.4 −1.8 −1.0 Prob 0.340 0.000 0.012 0.172 0.000— ANTROT CLN FUSERS DIPERS SOURST ECBDPE SCORE SCORE SCORE SCORE SCORE %NOT Stat ABS ABS ABS ABS ABS ABS Mean1 4.8 4.0 5.5 4.0 5.0 98.6 Mean23.7 5.0 6.3 3.5 3.0 95.9 Locs 6 1 6 3 1 1 Reps 10 3 10 6 1 1 Diff 1.1−1.0 −0.8 0.5 2.0 2.7 Prob 0.078 — 0.042 0.225 — — ECB1LF ECB2SC HSKCVRGIBROT DIPROT BRTSTK SCORE SCORE SCORE SCORE SCORE % NOT Stat ABS ABSABS ABS ABS ABS Mean1 4.3 4.8 6.1 3.5 2.5 98.8 Mean2 4.0 4.7 5.8 4.7 3.098.9 Locs 1 4 11 3 1 4 Reps 3 8 11 6 2 6 Diff 0.3 0.1 0.3 −1.2 −0.5 0.0Prob — 0.933 0.391 0.192 — 0.981 HD SMT ERTLPN LRTLPN % NOT % NOT % NOTStat ABS ABS ABS Mean1 86.6 93.6 83.6 Mean2 90.7 68.6 86.3 Locs 7 11 20Reps 11 14 23 Diff −4.1 25.0 −2.8 Prob 0.274 0.039 0.613

TABLE 5B INBREDS IN HYBRID COMBINATION REPORT Variety #1: HYBRIDCONTAINING PHACE Variety #2: 34B97 ESTCNT STKCNT ERTLPN COUNT COUNTPLTHT CM EARHT CM % NOT LRTLPN Stat % MN % MN % MN % MN ABS % NOT ABSMean1 104.5 100.8 93.1 96.6 100.0 100.0 Mean2 103.4 100.7 89.6 94.0100.0 90.0 Locs 2 10 1 1 2 1 Reps 4 20 2 2 4 2 Diff 1.1 0.1 3.5 2.5 0.010.0 Prob 0.764 0.895 — — 1.000 —

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All such publications, patents and patentapplications are incorporated by reference herein for the purpose citedto the same extent as if each was specifically and individuallyindicated to be incorporated by reference herein.

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding. Asis readily apparent to one skilled in the art, the foregoing are onlysome of the methods and compositions that illustrate the embodiments ofthe foregoing invention. It will be apparent to those of ordinary skillin the art that variations, changes, modifications and alterations maybe applied to the compositions and/or methods described herein withoutdeparting from the true spirit, concept and scope of the invention.

1. A seed comprising at least one set of the chromosomes of maize inbredline PHACE, representative seed of said line having been deposited underATCC Accession Number PTA-6341, wherein said chromosomes comprise all ofthe alleles of inbred line PHACE at the SSR loci listed in Table
 2. 2. Amaize plant produced by growing the seed of claim
 1. 3. A maize plantpart of the maize plant of claim
 2. 4. The seed of claim 1, wherein saidseed is an F1 hybrid maize seed produced by crossing a plant of maizeinbred line PHACE with a different maize plant and harvesting theresultant F1 hybrid maize seed.
 5. A maize plant produced by growing theF1 hybrid maize seed of claim
 4. 6. A maize plant part of the maizeplant of claim
 5. 7. An F1 hybrid maize seed comprising an inbred maizeplant cell of inbred maize line PHACE, representative seed of said linehaving been deposited under ATCC Accession Number PTA-6341.
 8. A maizeplant produced by growing the F1 hybrid maize seed of claim
 7. 9. The F1hybrid maize seed of claim 7 wherein the inbred maize plant cellcomprises two sets of chromosomes of maize inbred line PHACE, whereinsaid chromosomes comprise all of the alleles of inbred line PHACE at theSSR loci listed in Table
 2. 10. A maize plant produced by growing the F1hybrid maize seed of claim
 9. 11. A process of introducing a desiredtrait into maize inbred line PHACE comprising: (a) crossing PHACE plantsgrown from PHACE seed, representative seed of which has been depositedunder ATCC Accession Number PTA-6341, with plants of another maize linethat comprise a desired trait to produce F1 progeny plants, wherein thedesired trait is selected from the group consisting of waxy starch, malesterility, herbicide resistance, insect resistance, bacterial diseaseresistance, fungal disease resistance, and viral disease resistance; (b)selecting F1 progeny plants that have the desired trait to produceselected F1 progeny plants; (c) crossing the selected progeny plantswith the PHACE plants to produce backcross progeny plants; (d) selectingfor backcross progeny plants that have the desired trait and the allelesof inbred line PHACE at the SSR loci listed in Table 2 to produceselected backcross progeny plants; and (e) repeating steps (c) and (d)to produce backcross progeny plants that comprise the desired trait andcomprise at least 95% of the alleles of inbred line PHACE at the SSRloci listed in Table
 2. 12. A plant produced by the process of claim 11,wherein the plant comprises at least 95% of the alleles of inbred linePHACE at the SSR loci listed in Table
 2. 13. A maize plant having allthe physiological and morphological characteristics of inbred linePHACE, wherein a sample of the seed of inbred line PHACE was depositedunder ATCC Accession Number PTA-6341.
 14. A process of producing maizeseed, comprising crossing a first parent maize plant with a secondparent maize plant, wherein one or both of the first or the secondparent maize plants is the plant of claim 13, and harvesting theresultant seed.
 15. The maize seed produced by the process of claim 14.16. The maize seed of claim 15, wherein the maize seed is hybrid seed.17. A hybrid maize plant, or its parts, produced by growing said hybridseed of claim
 16. 18. A maize seed produced by growing said maize plantof claim 17 and harvesting the resultant maize seed.
 19. The maize plantof claim 13, further comprising an SSR profile in accordance with theprofile shown in Table
 2. 20. A cell of the maize plant of claim
 13. 21.The cell of claim 20, wherein said cell is further defined as having anSSR profile in accordance with the profile shown in Table
 2. 22. A seedcomprising the cell of claim
 20. 23. The maize plant of claim 13,further defined as having a genome comprising a single locus conversion.24. The maize plant of claim 23, wherein the single locus was stablyinserted into a maize genome by transformation.
 25. The maize plant ofclaim 23, wherein the locus is selected from the group consisting of adominant allele and a recessive allele.
 26. The maize plant of claim 23,wherein the locus confers a trait selected from the group consisting ofherbicide tolerance; insect resistance; resistance to bacterial, fungal,nematode or viral disease; yield enhancement; waxy starch; improvednutritional quality; male sterility and restoration of male fertility.27. The maize plant of claim 13, wherein said plant is further definedas comprising a gene conferring male sterility.
 28. The maize plant ofclaim 13, wherein said plant is further defined as comprising atransgene conferring a trait selected from the group consisting of malesterility, herbicide resistance, insect resistance, and diseaseresistance.
 29. A method of producing a maize plant comprising the stepsof: (a) growing a progeny plant produced by crossing the plant of claim13 with a second maize plant; (b) crossing the progeny plant with itselfor a different plant to produce a seed of a progeny plant of asubsequent generation; (c) growing a progeny plant of a subsequentgeneration from said seed and crossing the progeny plant of a subsequentgeneration with itself or a different plant; and (d) repeating steps (b)and (c) for an additional 0–5 generations to produce a maize plant. 30.The method of claim 29, wherein the produced maize plant is an inbredmaize plant.
 31. The method of claim 30, further comprising the step ofcrossing the inbred maize plant with a second, distinct inbred maizeplant to produce an F1 hybrid maize plant.
 32. A method for developing amaize plant in a maize plant breeding program using plant breedingtechniques wherein the plant breeding techniques comprise the step ofcrossing the maize plant of claim 13, or parts thereof, with a secondmaize plant.
 33. The method for developing a maize plant in a maizeplant breeding program of claim 32 wherein plant breeding techniques areselected from the group consisting of recurrent selection, backcrossing,pedigree breeding, restriction fragment length polymorphism enhancedselection, genetic marker enhanced selection, and transformation.
 34. Amethod of plant breeding comprising the steps of: (a) obtaining themolecular marker profile of maize inbred line PHACE, representative seedof said line having been deposited under ATCC Accession Number PTA-6341;(b) obtaining an F1 hybrid seed for which the maize plant of claim 13 isa parent; (c) crossing a plant grown from the F1 hybrid seed with adifferent maize plant; and (d) selecting progeny that retain themolecular marker profile of PHACE.